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
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • 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

Definitions

• compositions for mucosal administration are provided.
• the present invention relates to novel compositions for administration via the mucosa. More particularly, it relates to a pharmaceutical composition, in particular a vaccine composition intended for administration by the mucous route.
• It also relates to a method for producing antibodies, in particular IgA.
• Mucosal administration non-invasive administration of the antigen to a mucous site, for example
• Mucosal response response of the immune system to the mucous membranes characterized by production of IgA (isotype in high concentration in the mucous membranes) and of IgG and / or a cellular response within the mucous site and the draining glands.
• Svstemic response generalized immune system response resulting in the presence of circulating antibodies (IgG and also IgA) and / or a cellular response (Thelper or CTL) in secondary lymphoid organs (spleen, lymph nodes).
• Dissemination of the response installation of a mucosal response in other compartments than that of induction, due to the circulation and relocation of the induced lymphocytes to the site of administration (vaginal IgA after nasal administration for example).
• Adjuvant substance added to the antigen so as to amplify and orient the specific immune response of this antigen.
• Synthetic vector / vectorization inco oration of the antigen in or on the surface of a particle or vesicle, so as to protect the antigen and / or facilitate its capture by competent cells of the immune system and / or facilitate the preparation of the antigen by said cells, and thus to boost the immune response.
• the addition of an empty vector to the free antigen provides little or no amplification.
• mucous surfaces are considerable, on the one hand because they are present at the level of all the tracts and on the other hand because they are the first line of defense against the invasion of pathogenic agents.
• the protection of the mucous surfaces is ensured both by innate or non-adaptive defense mechanisms (peristalsis, ciliated movements and mucus) and by the implementation of an adaptive cellular and humoral immune response specific to the pathogen which can generalize to other lymphoid organs. It is the lymphoid tissue associated with the mucous membranes (MALT) which is responsible for the specific component.
• mucosal vaccination represents a considerable challenge .
• HIV hepatitis
• mucosal immunology For a fairly complete review of mucosal immunology, reference may be made to the "Handbook of mucosal immunology", edited by Pearay Ogra et al, 1994, Académie press, Inc. san Diego California, USA.
• liposomes - low stability (liposomes), - low efficiency (liposomes).
• Immunoglobulins or antibodies (Ac), associated with cellular components, are essential actors in the wild immune response against a pathogen.
• Ig are highly specific proteins of the antigen
• IgG In the blood compartment, the majority of the Ig produced are circulating IgG (subdivided into different subclasses), IgA and IgE remaining very minority. Conversely, the major isotype in the mucous membranes is IgA which is produced by the IgA plasma cells of the mucous lymphoid system and actively secreted by the epithehum of the mucous membrane.
• IgA are specialized antibodies adapted to the defense of mucous surfaces constantly exposed to pathogens Adapts because, because of their biochemical structure (glycosylation, polymerization, secretory), they resist the effects of proteases secreted by microorganisms and because, unlike other isotypes, they limit the inflammatory reactions in these compartments in a state of constant activation Specialized, because they act several secreted levels, they agglutinate pathogens, limit by neutralizing the effects of toxins and the entry of pathogens; they can also act within the epithelium during their transcytosis or in the lamina prop ⁇ a. They therefore represent an essential active barrier which, associated with non-specific mechanisms, limit invasion by pathogens. Naturally, with the antibody response, there are associated cytotoxic and specific cellular responses which aim to eliminate the pathogen. If the local response is not sufficient, a systemic response is triggered.
• parenteral administration of an antigen leads to the induction of specific circulating antibodies, of the IgG type. This type of injection does not make it possible to induce IgA within the mucous (or systemic) compartments. It is very clear that parenteral vaccination will not strengthen the local natural defenses necessary to fight against many respiratory or genital infections for example
• mucous membrane requires, in relation to parenteral administration, the management of the antigen in the mucosa and therefore the penetration of the antigen, which implies that the antigen must be optimally presented in order to overcome non-specific defense mechanisms (ciliated movements, mucus) and to resist enzymes present in the mucosa or on its surface. It appears that these parameters are optimized by the structure of the vesicles during mucosal administration. These functions of the vesicles in mucosal administration were not necessarily present during parenteral administration, where the injection makes it possible to directly reach areas more favorable for capture by the cells of the immune system.
• vesicles identical to those found in international application WO 99/16468 incorporating an antigen provoke an immune response much stronger than the administration of the free antigen
• their administration incorporated within multilamellar vesicles with onion structure, makes it possible to induce a quite remarkable response
• the induced response is found not only locally in the mucous compartment (response mucosa) but also in the blood circulation (systemic response)
• these multilamellar vesicles are prepared from biocompatible constituents known for their harmlessness.
• the preparation process is simple to implement and only uses common chemical devices. The fact that the process uses an initial lamellar phase at thermodynamic equilibrium gives it excellent reproducibility and allows great stability of the vesicles obtained
• the invention relates to the use of multilamellar vesicles with an onion structure having an internal liquid-crystal structure formed by a stack of concent ⁇ que bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which is found inco ⁇ or at least one antigen, for the manufacture of a composition, more particularly pharmaceutical, and in particular vaccine, intended for administration by mucous route.
• the antigen (s) are an integral part of the entity formed by the vesicle. Indeed, one can find molecules of antigen (s) in any layer between the center and the periphery of said vesicle.
• compositions used according to the invention allow the preparation of a mucous vaccine intended to induce a serum mucosal and or systemic response.
• the onion-structured vesicles as defined above inco ⁇ orating an antigen induce, when administered by mucosa, in humans or animals, the production of antico ⁇ s.
• one of the advantages of the invention is to induce a very large production of antico ⁇ s characterized by the presence of IgA and IgG. This implies an increase in the frequency of lymphocytes carrying IgA or IgG specific for the antigen.
• the preparation can therefore be used for activation and differentiation of antigen-specific B lymphocytes which can then be used in cell fusion in order to produce monoclonal antibodies.
• the specific lymphocytes present in large quantities in the ganglia draining the administration site can be immortalized by fusion with a non-secreting myeloma and lead to hybridomas secreting monoclonal antico ⁇ s.
• the invention by its capacity to amplify and increase the antico ⁇ s responses, can also be used for the purpose of producing antico ⁇ s in particular polyclonal IgA, isotype difficult to generate in conventional procedures, or polyclonal IgG .
• These antico ⁇ s can be used for non-therapeutic purposes, for example for research, more particularly for research in biology or immunology.
• the invention also relates to a process for producing antico ⁇ s. and more particularly of IgA. Furthermore, it has been demonstrated that the composition of the invention, when administered via the mucous membrane, induces protection of the organism with regard to the infection for which the antigen incorporated in said is responsible. composition.
• the invention relates to a method of treatment of human or animal co ⁇ s by vaccination by mucosa, according to which a composition containing multilamellar vesicles with onion structure having an internal c ⁇ stalquid structure formed is administered. of a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which there is inco ⁇ orated at least one antigen
• compositions of the invention in which the antigen is found inco ⁇ orated in a vesicle with onion structure, as defined above allow, when 'They are administered via the mucosa, to elicit a mucosal immune response and to stimulate the lymphoi ' tissue of common mucosa.
• Such capacities prove to be of particular importance when dealing with antigens with vaccinating potential, in the face of mvasive pathogens with mucous tropism.
• the results obtained in the context of the invention demonstrate that the invention not only makes it possible to amplify the antico ⁇ s response, but that it also generates an immune response with protective efficacy against infection.
• the invention is therefore applicable both to the production of antico ⁇ s, and to vaccination
• the results obtained make it possible to affirm that the vesicles administered by mucous route, in particular by nasal route, can be used in order to induce or amplify the antico ants response in the mucous compartments, to disseminate this response to other sites more away from the administration site and generate a systemic response
• the vesicles used according to the invention generally have diameters between 0.1 and 25 ⁇ m, preferably between 0.2 and 15 ⁇ m.
• the vesicles used according to the invention preferably consist of several layers of amphiphilic agents alternating with layers of aqueous or polar phase.
• the thickness of each of these layers is a molecular thickness, typically of the order of 5 to 10 nanometers.
• a comp ⁇ s diameter between 0.1 ⁇ m and a few tens of micrometers. This is what is observed experimentally, the vesicles being observable by optical microscopy (in polarized light in order to have a better contrast linked to their birefringence), either as unresolved points for the smallest of them, or as birefringent spheres for larger ones.
• the size profile can be studied using a laser granulometer (using the static scattering of a laser beam, analyzed from several angles).
• a laser granulometer using the static scattering of a laser beam, analyzed from several angles.
• the vesicles in which the antigen is found have, as previously explained, a multilamellar onion structure and are made up, from their center to their periphery, of a succession of lamellar layers separated by a liquid medium. These vesicles can be obtained by a process comprising the preparation of a crystalline lamellar phase and its transformation by application of a shear. Such a method is in particular described in patent WO 93/19735 from French patent FR-2 689 418 or WO 95/18601 introduced here by reference.
• Such vesicles have, among other things, the advantage of being able to be prepared by a particularly simple preparation process making it possible to use a large variety of surfactants.
• Another advantage hey also essentially to the process used to prepare the onion-structure vesicles used according to the invention, lies in the fact that the ingredients and the additives are incorporated prior to the formation of the vesicles, which allows a excellent encapsulation yield, hence better efficiency and very significant savings for extremely expensive molecules.
• Such structures are advantageously obtained by inco ⁇ oration of at least one antigen in a c ⁇ stalquide lamellar phase comprising at least one surfactant, then transformation of this lamellar c ⁇ stalquide phase into a dense phase of small multilamellar vesicles.
• the vesicles used according to the invention can be obtained according to a process according to which a lamellar crystal-liquid phase is incorporated which inco ⁇ or at least one antigen and the said crystal-liquid phase is reacted in multilamellar vesicles by application of a shear
• This shear pou ⁇ a be a homogeneous shear, which has the advantage of leading to vesicles of perfectly homogeneous size.
• simple mechanical stirring may prove to be sufficient to lead to the formation of the multilamellar vesicles of the invention.
• the antigen may be any molecule for which it is desired to generate an immune response, whether it has an exogenous source such as an infectious pathogenic organism, parasites or microorganisms (yeasts, fungi, bacteria or viruses) or an intrinsic natural origin. (case of autoimmune diseases or cance ⁇ sation) It can be of different biochemical natures. It can, in particular, be an antigen chosen from the group consisting
• the multilamellar vesicles with onion structure are prepared according to the methods previously described, in particular in international application WO 99/16468
• the amphiphilic molecules used for their preparation will be chosen, without this being an obligation, from among the molecules described in the pharmacopoeia, or already used in drugs applied to the mucous membranes.
• the membranes of the vesicles contained in the compositions of the invention contain at least one surfactant chosen from the group consisting of:
• - Cg to C30 fatty acids saturated or mono- or polyunsaturated, linear or branched, in the form of the acid or salt of an alkali, alkaline earth metal or of an amine, - esters, ethoxylated or not , of these same fatty acids and
• - C 1 to C 30 fatty alcohols saturated or mono- or polyunsaturated, linear or branched, ethoxylated or not, - ethers, ethoxylated or not, of these same fatty alcohols and
• - copolymers of polyethylene glycol and of alkyl glycol for example the family of ELFACOS from AKZO NOBEL
• - di- or triblock copolymers of ethers of polyethylene glycol and of polyalkylene glycol for example the family of ARLACELL from ICI
• Co-surfactants may be added to these surfactants which can be used alone or as a mixture in order to improve the viscosity and the tightness of the membranes forming the vesicle.
• these molecules there may be mentioned: - cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesterol sulfate
• the formulation advantageously involves a mixture of surfactant molecules. It is generally used at least two different surfactants having different hydrophilic-lipophilic scales, which makes it possible to continuously adjust the properties of the bilayers and thus to control the appearance of the instability which governs the formation of multilamellar vesicles.
• two surfactants having relatively different properties in particular a different hydrophilic-lipophilic balance (HLB).
• the first surfactant will advantageously have a hydrophilic-lipophilic balance comp ⁇ se between 1 and 6, preferably between 1 and 4, while the second surfactant will have a hydrophilic-lipophilic balance between 3 and 15, preferably between 5 and 15
• the preparation obtained after transformation of the c ⁇ stal-hquide lamellar phase into multilamellar vesicles can then be diluted, in particular with an aqueous solvent such as. for example, a buffer solution, a saline solution or a physiological solution, thereby obtaining an aqueous suspension of vesicles
• an aqueous solvent such as. for example, a buffer solution, a saline solution or a physiological solution
• the encapsulation technique used according to the present invention makes it possible to easily achieve very high encapsulation yields, or even close to 100%. However, such yields are not always essential depending on the intended applications.
• the encapsulation yield of the antigen (s) in the compositions of the invention is advantageously greater than 50%, preferably greater than 80%
• the structure of the vesicles is responsible for the particularly advantageous results obtained, and that the multilamellar vesicles of the invention allow the antigen to arrive intact up to the antigen presenting cells (APC) and to promote its capture. by these cells. So it looks good that the function of the vesicles of the invention is to vectorize, protect and improve the capture of the antigen by the immune system.
• APC antigen presenting cells
• Another advantage of the technology is that we can add to this formulation natural or artificial polymers such as polysaccha ⁇ des (alginates, chitosan, etc.) in order to strengthen the solidity of the vesicle, and allow it to stay longer on the site of administration or in the organism, thus delivering the antigen over a longer time.
• These polymers can be incorporated in the vesicle as well as deposited around it in the form of a coating.
• the vesicle or the particle formed from vesicles coated in the polymeric mat ⁇ ce has a diameter greater than that of the vesicles alone.
• These polymers can optionally be crosslinked to further strengthen their solidity.
• the formulation can be supplemented by the addition of immunomodulating molecules (chitosan, interleukmes 7) which will reinforce by their intrinsic properties the amplification and the orientation of the immune response.
• immunomodulating molecules chitosan, interleukmes
• the vesicles inco ⁇ orating the antigens are advantageously prepared in a process consisting in preparing, firstly, the lamellar phase. This is obtained by simple mixing of the ingredients, in an order determined by the experimenter according to the miscibihtees of each of the constituents. It may be necessary to heat certain pasty or solid constituents in order to facilitate their inco ⁇ oration.
• the antigen is added preferably at the end of the mixture in order to avoid it being subjected to an excessively high temperature. It is also possible to prepare a mixture of all the constituents except for the antigen or its aqueous solution in the form of a “stock” mixture which will be used as necessary to prepare the lamellar phase.
• the aqueous solution can contain different constituents intended to ensure its biological compatibility and in particular buffer mixtures but also different antigens.
• the lamellar phase thus prepared is then subjected to moderate shearing (from 0 to 1000 s ⁇ 1 ) for a limited time (from 0 to 60 minutes).
• this shearing is obtained directly by the action of the device used for mixing.
• it can be obtained by hand by mixing the preparation using a microspatula in an Eppendorf type tube.
• the sheared lamellar phase is then dispersed in a final medium, in the form of water. or a stamp, identical or different from that used during of the preparation of the lamellar phase.
• This dispersion is advantageously carried out at room temperature (20-25 ° C) by slow addition of the medium to the lamellar phase with constant stirring.
• a preservative and possibly other additives intended to complete the galenical formulation can be added to the product.
• compositions previously determined comprising at least one inco ⁇ orated antigen within the vesicles with a lamellar structure onion have the advantage of being able to be used for administration by mucous route and very particularly by nasal route and of inducing a mucosal response and / or systemic.
• Figures 1 and 2 given with reference to this example bring together the responses obtained on the one hand in different mucous samples (Figure 1) and on the other hand in the sera of animals (group II) in comparison with those obtained by administration either of compositions in which the same antigen is free (group II), or of compositions containing the same but empty vesicles (group III).
• Example II illustrates a method of immunization with FHA. The results obtained in this example are illustrated by Figures 3, 4 and 5 which respectively show
• HSA human serum albumin
• the components are sterilized by UV irradiation for 60 min.
• Containers and accessories (spatulas, stirrers ...) are sterilized with the flame just prior use.
• the constituents O to ⁇ are introduced into a piluher, in an indifferent order, then heated at 80 ° C. for 60 min with very strong magnetic stirring. The total dissolution of the constituents ⁇ and ⁇ is checked by microscopic observation.
• the preparation is then dispersed at 33.33% in sterile 1x PBS.
• the sou ⁇ s were divided into 4 groups noted I to IV, group IV constituting a control group (unimmunized mice also called naive mice) and the other groups being subjected to an immunization protocol as defined above using the following products • - group I:
• Encapsulated HSA 20 ⁇ l per nostril of vesicles according to the invention inco ⁇ orating HSA which corresponds to 80 ⁇ g of HSA per mouse
• mice The trachea of the mice is cannulated using a probe and 750 ⁇ l of PBS are injected slowly so as not to create hemorrhage, the lungs are thus washed 3 times with the same solution, the sample is then centrifuged in order to remove lung cells and separate into aliquots kept at -20 ° C until assay.
• Intestinal washes The intestine is removed, released from the mesentery and rinsed in water to remove outside blood. It is then cut longitudinally and incubated on ice in 1 ml of a washing solution enriched in protease inhibitor. The whole is centrifuged and the supernatant is collected and frozen at -20 ° C.
• the specific HSA antico danss present in the sera and secretions are assayed by ELISA technique in which the IgA and IgG specific for HSA are determined (biotinylated anti-IgA / Streptavidin peroxidase, anti-IgG peroxidase).
• the results are expressed in mean titer determined relative to a reference serum of naive mouse and corresponding to the inverse of the dilution equal to the reference threshold.
• the specific isotype response of HSA predominant in the intestine and the vagina is IgA.
• IgG H + L titration of IgG H + L could not be performed in vaginal lavage, experiments similar tests performed with another antigen indicate the predominance of IgA in vaginal secretions.
• the FHA protein is a filamentous adhesin from Bo detella pertussis. pertussis bacteria Unlike HSA previously used, FHA is more immunogenic and therefore capable of inducing an antico ⁇ s response by itself This protein is one of the protective antigens contained in commercial pertussis vaccines Finally, the mu ⁇ n model can be infects with Bordetella pertussis and it is thus possible to carry out an infection test following mucosal immunization and to show the protective character of the response induced by the immunization A - Induction of antibodies
• the operating procedure for the preparation is similar to that of Example 1.
• the finished product has an FHA concentration of 3 ⁇ g per 40 ⁇ l.
• the mucosal immunization and sampling protocol is identical to what was described in Example 1. Two groups of 5 animals were formed, the group I receiving the antigen encapsulated in the vesicles, and group II receiving the non-encapsulated antigen, in solution in PBS. At each immunization, each animal receives 3 ⁇ g of FHA, divided into two instillations, one in each nostril.
• the specific FHA antico ⁇ s present in the serums and secretions are doses by ELISA technique according to a protocol identical to that dec ⁇ t in Example 1, with the reagents specific to the FHA antigen.
• FIGS. 3 and 4 The results are presented in FIGS. 3 and 4, where the mean titers of IgA and IgG antico ⁇ s in the mucous secretions (FIG. 3) and in the serum (FIG. 4) are given for the two groups I and II.
• the mucous secretions are respectively studied as in Example I, from broncho-alveolar washes (A), intestinal washes (B) and vaginal washes (C).
• mice immunized with the antigen incorporated in the vesicles of the invention (FIG. 3) all of the animals respond to the antigen whereas when the free antigen is administered, only 1/5 of the animals are responders and this in a very weak way.
• results of this example 2 completely confirm those obtained on HSA. They make it possible to affirm that the vesicles administered by the nasal route can be used in order to induce or amplify the antico ⁇ s response in the mucous compartments. disseminate this response to other sites further away from the administration site and generate a systemic response.
• the formulation and the preparation of the vesicles are stnctement identical to that previously used for the caracté ⁇ sation of the response antico ⁇ s.
• mice Three groups of mice are used:
• mice Sou ⁇ s immunized with the inco ⁇ orated antigen in the vesicles of the invention IL Mice immunized with free antigen in solution in PBS III. Unimmunized mice
• Each group consists of 4 mice, immunized at the same time, with the same batch of antigen, in order to be able to carry out all the infection measurements under the same conditions.
• mice Four weeks after the second immunization, the mice are anesthetized and infected with Bordetella pertussis by the nasal route (5.10 7 bacteria / mice administered in 20 ⁇ l) and the initial infectious load is checked as early as 3 hours after infection.
• mice are sacrificed and their lungs are removed at various times after infection and diluted in PBS until a homogeneous suspension is obtained. Different dilutions of the suspension are spread on a specific nutet medium of Bordetella and the colonies are counted after three days of growth.
• mice immunized with the inco incorated antigen in vesicles of the invention mice immunized with the inco incorated antigen in vesicles of the invention.
• Group II mice immunized with free antigen in solution in
• Group III non-immunized mice.
• mice immunized with the antigen inco ⁇ oré in the vesicles of the invention have a lower bacterial load (factor of 2) than that of mice immunized with the free antigen, and much lower than that of unimmunized mice (factor 8).
• factor 2 the bacterial load
• the mice immunized with the free antigen as well as the non-immunized mice see their bacterial load increase, the mice immunized with the incororated antigen in the vesicles of the invention have fewer bacteria on D5 than on D3 indicating a favorable evolution of the disease.
• the number of bacterial colonies in the group immunized with the inco ⁇ orated antigen in the vesicles of the invention is 6 times lower than in the group immunized with free FHA and 13 times lower than in the group not immune indicating good protection.

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• 1999
  • 1999-09-14 FR FR9911465A patent/FR2798288B1/fr not_active Expired - Fee Related
• 2000
  • 2000-09-13 WO PCT/FR2000/002523 patent/WO2001019335A2/fr active IP Right Grant
  • 2000-09-13 AU AU74284/00A patent/AU782653B2/en not_active Ceased
  • 2000-09-13 JP JP2001522970A patent/JP2003509352A/ja active Pending
  • 2000-09-13 EP EP00962621A patent/EP1212043A2/de not_active Withdrawn
  • 2000-09-13 CA CA002384876A patent/CA2384876A1/en not_active Abandoned

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