Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • 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
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
• • 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
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • 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
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

• the present invention relates to new adjuvants for vaccine compositions as well as compositions comprising at least one antigen, in particular an antigen of viral, bacterial or parasitic origin and at least one adjuvant.
• the development of vaccines inactivated or containing purified antigens is becoming increasingly important, as it makes it possible to avoid undesirable side effects.
• improving the quality of antigens comes at the expense of their immunogenic character. It is for this reason that they are associated with immunity adjuvants.
• Immunity adjuvants are products that increase the reactions of the immune system when administered in the presence of antigens of viral, bacterial or synthetic origin. They cause the massive appearance of macrophages at the injection site, then in the lymphatic nodules, increase the production of specific immunoglobulins, antibodies, and stimulate many cells involved in the mechanisms of immune defense. These adjuvants are of various natures. They can for example consist of liposomes or emulsions.
• Very effective Freund's adjuvants result from the association of a mineral oil and a mannitol ester containing or not containing a killed mycobacterium.
• the vaccines produced by mixing in equal parts a Freund's adjuvant with an aqueous antigenic medium still remain the benchmarks worldwide for laboratory studies. They are in the form of water in oil emulsions (W / O), that is to say emulsions in which the continuous phase is oil.
• W / O oil in oil emulsions
• the active ingredient is generally located in the aqueous phase, which is often a buffered saline solution. This phase is in the form of drops separated from each other by an oily film. This formulation makes it possible to obtain amplified and prolonged biological responses over time.
• emulsions of this type are generally very viscous and are therefore difficult to inject. They often require the use of syringes with a large needle diameter and are the cause of pain during the injection and trauma at the injection site.
• the aqueous phase content of the injectable W / O emulsions is around 30
• a W / O type emulsion will be called fluid, if its viscosity is less than 4 times that of this reference emulsion, that is to say less than 500 mPa.s at 25 ° C (BROOKFIELD LVT M 2 V 30 ).
• fluid emulsions are generally less stable than more viscous emulsions, since phase shifts are observed only a few days after their preparation.
• the Applicant has therefore sought to develop emulsions of the water-in-oil type (W / O), concentrated in the aqueous phase which do not have the drawbacks set out above, that is to say which are fluid and stable. over time.
• the stability criterion of the emulsion being considered satisfied if it does not present any major defect after 1 month at the following three temperatures: 4 ° C, 25 ° C and 37 ° C.
• the subject of the invention is a composition comprising a fatty phase H and an aqueous phase E, in the form of an emulsion of the water-in-oil type and characterized in that the weight ratio W / O is greater than 1.
• the weight ratio E is a composition comprising a fatty phase H and an aqueous phase E, in the form of an emulsion of the water-in-oil type and characterized in that the weight ratio W / O is greater than 1.
• the fatty phase constituting the composition which is the subject of the present invention generally comprises one or more compounds chosen from oils of mineral, vegetable or animal origin, the alkyl esters of said oils, the alkyl esters of fatty acids or the alkyl ethers of fatty acids, esters of fatty acids and polyols or ethers of fatty alcohols and polyols.
• oils of mineral origin there are oils of petroleum origin, such as white mineral oils such as MARCOL TM 52 or DRAKEOL TM 6 VR.
• vegetable oils there are peanut oil, olive oil, sesame oil, soybean oil, wheat germ oil, oil grape seed, sunflower oil, castor oil, linseed oil, soybean oil, corn oil, copra oil, palm oil, oil nuts, hazelnut oil, rapeseed oil or squalane or olive squalene.
• oils of animal origin are spermaceti oil, tallow oil, squalane or squalene extracted from fish livers.
• alkyl esters of oils are the ethyl, linear or branched propyl or butyl, linear or branched methyl esters of said oils.
• fatty acids suitable for the preparation of the esters mentioned above there are more particularly, those containing from 12 to 22 carbon atoms, such as for example, myristic acid, palmitic acid, oleic acid, ricinoleic acid or isostearic acid and advantageously a fatty acid liquid at 20 ° C.
• fatty acid esters or fatty acid ethers are the alkyl esters of fatty acids, such as, ethyl oleate, methyl oleate, isopropyl myristate or octyl palmitate, esters of fatty acids and polyols or ethers of fatty alcohols and polyols, such as fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, fatty acid esters with a polyglycerol or fatty acid esters of propylene glycol, and more particularly fatty acid esters with a hexol, such as for example sorbitol or mannitol, fatty acid esters with a hexol anhydride, such as sorbitan or mannitane.
• alkyl esters of fatty acids such as, ethyl oleate, methyl oleate, isopropyl myristate or octyl
• the fatty phase may comprise only one of the compounds mentioned above or else a mixture of several of the compounds mentioned above.
• the fatty phase constituting the composition which is the subject of the present invention, also comprises one or more surfactants having an overall HLB number of between 5 and 8.
• the fatty phase comprises approximately between 1% and 15% by weight, preferably between 3 % and 10% by weight of surfactants.
• the HLB of the mixture is the weighted sum of the HLBs of each surfactant.
• a more particular subject of the invention is a composition as defined above in which the surfactant or the mixture of surfactants has an overall HLB number is greater than or equal to 6 and less than 8.
• the weight ratio aqueous phase on fatty phase W / O is greater than 3/2.
• the surfactants used are generally chosen from modified fatty substances.
• modified fatty substances used in the context of the present invention can be of vegetable or animal mineral origin.
• modified fats of mineral origin there are oils of petroleum origin.
• modified fatty substances of vegetable origin there are modified vegetable oils, for example, modified oils of peanut, olive, sesame, soy, wheat germ, sunflower grape seeds, castor, flax, soy, corn, copra, palm, nuts, hazelnuts or rapeseed.
• modified fatty substances of animal origin are modified squalane, modified squalene, modified spermaceti oil or modified tallow oil.
• modified fatty substances denotes in particular the alkoxylated derivatives of fatty substances and more particularly the alkoxylated derivatives of oils or the alkoxylated derivatives of alkyl esters of oils and more particularly, the ethoxylated and / or propoxylated derivatives of oils or the ethoxy and / or propoxylated derivatives of the methyl, ethyl, linear or branched propyl or butyl, linear or branched esters of said oils.
• a more specific subject of the invention is a composition as defined above, in which the modified fatty substance is chosen from ethoxylated derivatives of oils having a number of EOs, between 1 and 10.
• modified fatty substances also denotes esters of fatty acids and of polyols or ethers of fatty alcohols and of polyols, and more particularly, esters of fatty acids with a hexol, such as for example sorbitol or mannitol or esters of fatty acids with a hexol anhydride, such as sorbitan or mannitane, alkoxylated derivatives of fatty acid esters and of polyols or alkoxylated derivatives of fatty alcohol ethers and of polyols, like the triglycerides of alkoxylated fatty acids, the alkoxylated esters of polygly- fatty acid cerol, and more particularly the alkoxylated esters of fatty acids with a hexol, such as for example sorbitol or mannitol or the alkoxylated esters of fatty acids with a hexol anhydride, such as sorbitan
• esters of fatty acids and polyols is meant in the context of the present invention, monoesters of fatty acids and polyols or polyesters of fatty acids and polyol such as for example diesters of fatty acids and polyols or triesters of fatty acids and polyols. It is the same for the polyalkoxylated derivatives of said esters.
• ethers of fatty acids and polyols is meant in the context of the present invention, monoethers of fatty acids and polyols or polyethers of fatty acids and polyol such as for example acid diethers fatty acids and polyols or triethers of fatty acids and polyols. It is the same for the polyalkoxylated derivatives of said ethers.
• a more particular subject of the invention is a composition as defined above, in which the modified fatty substances are chosen from the ethoxy derivatives of fatty acid esters and of polyols or the ethoxy derivatives of ethers of fatty alcohols and of polyols, and more particularly, the ethoxyl esters of fatty acids with a hexol, such as for example sorbitol or mannitol or the ethoxyl esters of fatty acids with a hexol anhydride, such as sorbitan or mannitane having a number of EOs, between 5 and 10.
• the modified fatty substances are chosen from the ethoxy derivatives of fatty acid esters and of polyols or the ethoxy derivatives of ethers of fatty alcohols and of polyols, and more particularly, the ethoxyl esters of fatty acids with a hexol, such as for example sorbitol or mannitol or the
• fatty acids suitable for the preparation of the modified fatty substances described above there are those containing on average from 12 to 22 carbon atoms, such as for example, those comprising 16 to 18 carbon atoms, such as oleic acid, ricinoleic acid or isostearic acid and advantageously fatty acids which are liquid at 20 ° C.
• the surfactant or the mixture of surfactants present in the fatty phase essentially consists of one or more esters chosen from mannitane esters, sorbitan esters, mannitane esters alkoxylated or alkoxylated sorbitan esters.
• the surfactant present in the fatty phase consists of a mixture of mannitane oleate and polyethoxylated mannitane oleate, a mixture of sorbitan oleate and polyethoxylated sorbitan oleate, a mixture of sorbitan oleate and polyethoxylated mannitane oleate or a mixture of mannitane oleate and polyethoxylated sorbitan oleate, said mixtures having an overall HLB number is greater than or equal to 6 and less than 8.
• composition according to the invention can also include one or more conventional immune stimulating agents, such as Avridine TM, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine, derivatives of MDP ( muramyl dipeptide), in particular threonyl-MDP, mycolic acid derivatives or lipid A derivatives.
• the composition according to the invention optionally comprises one or more organic salts of water-soluble metal cations, such as, for example, gluconate of calcium, manganese gluconate aluminum salicylate or soluble aluminum acetate.
• the adjuvant composition according to the invention comprises a pharmaceutically acceptable salt
• the latter is at a concentration of 0.02 to 3000 mg / cm 3 , preferably 0.1 to 1000 mg / cm 3 , more preferably from 0.1 to 150 mg / cm 3 .
• composition according to the invention may also comprise one or more insoluble salts generally used as immunity adjuvants, such as aluminum hydroxide or calcium phosphate.
• compositions as defined above further comprising, at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence and a non-zero amount of a composition as defined above.
• the term “antigen or at least one in vivo generator of a compound comprising an amino acid sequence” denotes either killed microorganisms, such as viruses, bacteria or parasites, or purified fractions of these microorganisms. , or living microorganisms whose pathogenic power has been reduced.
• viruses which can constitute an antigen according to the present invention there is the rabies virus, herpes viruses, such as Aujeszky's disease virus, orthomixoviruses such as Influenzae, picornaviruses such as the FMD virus or retroviruses such as HIV.
• viruses which can constitute an antigen according to the present invention there is the rabies virus, herpes viruses, such as Aujeszky's disease virus, orthomixoviruses such as Influenzae, picornaviruses such as the FMD virus or retroviruses such as HIV.
• E a microorganism of the bacterial type which can constitute an antigen according to the present invention.
• Coli and those of the genera Pasteurella, Furonculosis, Vibriosis, Staphylococcus and Streptococcus.
• parasites are those of the genera Trypanosoma, Plasmodium and Leishmania.
• Mention may also be made of recombinant viruses, in particular non-enveloped viruses, such as adenoviruses, vaccinia virus, Canarypox virus, herpes viruses or baculoviruses.
• a non-enveloped living viral recombinant vector is also designated, the genome of which contains, preferably inserted into a part which is not essential for the replication of the corresponding enveloped virus, a sequence coding for an antigenic subunit inducing antibody synthesis and / or a protective effect against the above enveloped virus or pathogenic microorganism; these antigenic subunits can be, for example, a protein, a glycoprotein, a peptide or a peptide fraction and / or protective against infection by a living microorganism such as an enveloped virus, bacteria or parasite.
• the exogenous gene inserted into the microorganism can, for example, be derived from an Aujeszky or HIV virus.
• the aim of this last nucleotide sequence is to allow the expression of a compound comprising an amino acid sequence, this compound itself having the aim of triggering an immune reaction in a host organism.
• the term “in vivo” generator of a compound comprising an amino acid sequence means a whole biological product capable of expressing said compound in the host organism into which said generator has been introduced in vivo.
• the compound comprising the amino acid sequence can be a protein, a peptide or a glycoprotein.
• generators in vivo are generally obtained by methods resulting from genetic engineering. More particularly, they can consist of living microorganisms, generally a virus, playing the role of recombinant vector, into which is inserted a nucleotide sequence, in particular an exogenous gene. These compounds are known as such and used in particular as a recombinant unitary vaccine. In this regard, reference may be made to the article by M. ELOIT et al., Journal of virology (1990) 71, 2925-2431 and to the international patent applications published under the numbers WO-A-91/00107 and WO -A-94/16681.
• the in vivo generators according to the invention can also consist of a recombinant plasmid comprising an exogenous nucleotide sequence, capable of expressing in a host organism a compound comprising an amino acid sequence.
• a recombinant plasmid comprising an exogenous nucleotide sequence, capable of expressing in a host organism a compound comprising an amino acid sequence.
• Such recombinant plasmids and their mode of administration in a host organism were described in 1990, by LIN et al., Circulation 82: 2217,2221; COX et al., J. of VIROL, Sept. 1993, 67, 9, 5664-5667 and in the international application published under the number WO 95/25542.
• the compound comprising the amino acid sequence which is expressed within the host organism can: (i) be an antigen, and allow the triggering of a immune reaction,
• (ii) have a curative action vis-à-vis a disease, essentially a functional disease, which is triggered in the host organism.
• the in vivo generator allows treatment of the host, of the gene therapy type.
• such a curative action may consist in a synthesis by the in vivo generator of cytokines, such as interleukins, in particular interleukin 2.
• cytokines such as interleukins, in particular interleukin 2.
• Cel- these allow the triggering or strengthening of an immune reaction aimed at the selective elimination of cancer cells.
• the vaccine composition as defined above comprises a concentration of antigen which depends on the nature of this antigen and on the nature of the subject treated. It is however particularly remarkable that an adjuvant according to the invention makes it possible to significantly reduce the usual dose of antigen required.
• the appropriate concentration of antigen can be determined conventionally by those skilled in the art. Generally, this dose is of the order of 0.1 ⁇ g I cm 3 to 1 g / cm 3 more generally between 1 ⁇ g / cm 3 and 100 mg / cm 3 .
• the concentration of said generator in vivo in the composition according to the invention depends, in particular, on the nature of said generator and on the host in which it is administered. This concentration can be easily determined by a person skilled in the art, on the basis of routine experience.
• the in vivo generator when the in vivo generator is a recombinant microorganism, its concentration in the composition according to the invention generally between 10 2 and 10 15 microorganisms / cm 3 and preferably between 10 5 and 10 12 microorganisms / cm 3 .
• the generator in vivo when the generator in vivo is a recombinant plasmid, its concentration in the composition according to the invention can be between 0.01 g / dm 3 and 100 g / dm 3 .
• the vaccine as defined above, is prepared by mixing the adjuvant phase and the antigenic phase, optionally adding water or a pharmaceutically acceptable diluent medium.
• the invention preferably relates to W / O type emulsions as defined above, which are fluid.
• fluid emulsion we mean emulsions whose viscosity, measured at 25 ° C with a BROOKFIELD LVT viscometer with rotating mobile (2 speed mobile 30 rpm) is less than 500 mPa.s (BROOKFIELD LVT M 2 V 30).
• the vaccine composition as described above can be used as a preventive or curative medicine.
• a composition according to the invention can be administered to fish, crustaceans such as shrimps, poultry, in particular, geese, turkeys, pigeons and chickens, canines such as dogs, felines such as cats, pigs, primates, bovines, ovids and horses.
• the composition according to the invention can also be administered to humans.
• the administration of the composition can be carried out in a conventional manner by the parenteral route, in particular by subcutaneous, intramuscular or intraperitoneal injection. It can also be done orally or nasally.
• the subject of the invention is a composition essentially consisting of 1% and 15% by weight, and preferably 3% and 10% by weight, of surfactants having an HLB number overall between 5 and 8, and from 85% to 99 %, and preferably from 90% to 97% by weight, of one or more compounds chosen from oils of mineral, vegetable or animal origin, the alkyl esters of said oils, the alkyl esters of fatty acids or the alkyl ethers fatty acid esters of fatty acids and polyols or ethers of fatty alcohols and polyols.
• the oil is more particularly chosen from white mineral oils.
• the surfactant or the mixture of surfactants preferably has an overall HLB number greater than or equal to 6 and less than 8.
• the surfactant or the mixture of surfactants are chosen from modified fatty substances.
• modified fatty substance denotes in particular the alkoxylated derivatives of oils or the alkoxylated derivatives of alkyl esters of oils and more particularly, the ethoxylated and / or propoxylated derivatives of oils or the ethoxylated and / or propoxylated derivatives of methyl esters , ethyl, linear or branched propyl or butyl, linear or branched, of said oils and more particularly from the ethoxy derivatives of oils having a number of EOs, between 1 and 20, preferably between 5 and 10.
• modified fatty substance also denotes esters of fatty acids and of polyols or ethers of fatty alcohols and of polyols, and more particularly, esters of fatty acids with a hexol, such as for example sorbitol or mannitol or esters of fatty acids with a hexol anhydride, such as sorbitan or mannitane, alkoxylated derivatives of fatty acid esters and of polyols or alkoxylated derivatives of fatty alcohol ethers and of polyols, and more particularly, the alkoxylated esters of fatty acids with a hexol, such as for example sorbitol or mannitol or the alkoxylated esters of fatty acids with a hexol anhydride, such as sorbitan or mannitane having a number EO, between 1 and 20, more particularly, the ethoxyl derivatives of fatty acid esters and of
• the fatty acids suitable for the preparation of modified fatty substances preferably contain on average from 12 to 22 carbon atoms and more particularly 16 to 18 carbon atoms.
• the surfactant or the mixture of surfactants are derivatives of oleic acid.
• the surfactant or surfactants are chosen from mannitane esters, sorbitan esters, alkoxylated mannitane esters or esters of alkoxylated sorbitan.
• the oily composition as defined above contains as surfactant system, a mixture of mannitane oleate and ethoxylated mannitane oleate having a number of EOs, between 5 and 10.
• its subject is a process for the preparation of a water-in-oil type vaccine emulsion, as defined above; comprising the addition in an W / O weight ratio greater than 1, of an aqueous medium containing at least one antigen or at least one in vivo generator of a compound comprising a sequence of amino acids to the oily composition as defined above, as well as the use of said oily composition as an adjuvant in injectable compositions such as vaccines.
• the process as defined above can be carried out cold, that is to say at a temperature between 0 ° C and 30 ° C.
• An emulsion is prepared according to the invention, by pouring an aqueous phase consisting of a saline solution of physiological saline, onto a fatty phase, with stirring with a high shear mixer (SILVERSON L4RT). 200 g of emulsion are prepared for each test, the aqueous phase representing 65% of the weight of the emulsion.
• the fatty phase consists of:
• the viscosity of the emulsion is checked after 24 hours of stabilization at 25 ° C (BROOKFIELD LVT M 2 V 30). It is equal to 430 mPa.s.
• the sample obtained is divided into three bottles for evaluation of the stabilities at the following three temperatures: 4 ° C, 25 ° C and 37 ° C. It is found that this emulsion is stable at the three temperatures tested. After 1 month of storage, the emulsion is not broken and the oil release is less than 5%.
• Example 2 An emulsion according to the invention is prepared as described in Example 1, in which the aqueous phase represents 65% by weight thereof, and in which the fatty phase consists of
• the viscosity of the emulsion is checked after 24 hours of stabilization at 25 ° C. It is equal to 134mPa.s. (BROOKFIELD LVT M 2 V 30). The sample obtained is divided into three bottles for evaluation of the stabilities at the three temperatures (4 ° C / 25 ° C / 37 ° C).
• Example 1 An emulsion according to the invention is prepared as described in Example 1, in which the aqueous phase represents 65% by weight thereof, and in which the fatty phase consists of:
• the viscosity of the emulsion is checked after 24 hours of stabilization at 25 ° C. It is equal to 360 mPa.s. (BROOKFIELD LVT M 2 V 30).
• the sample obtained is divided into three bottles for evaluation of the stabilities at the three temperatures (4 ° C / 25 ° C / 37 ° C).
• this emulsion is stable at the three temperatures tested. After one month of storage, the emulsion is not broken and the oil release is less than 5%.

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