FR2803517A1 - AMPHIPHILIC AND IONIC PLYMERIC MATRICES AND DERIVATIVES THEREOF - Google Patents

Abstract

The invention relates to a novel type of amphilic and ionic polymer matrixes comprising a macromolecular hydrophilic matrix bearing a positive or negative ionic charge, whereby a lipidic phase having a sign opposite to that of the matrix is incorporated therein. The invention also refers to a method for the production and use thereof.

Description

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 The present invention relates to a new type of polymer matrices characterized in that they carry a positive or negative charge and in which is incorporated a lipid phase of charge opposite to that of the matrix. The invention also relates to their method of manufacture and their use.

 The search for new drug delivery systems is now indispensable for the development of new pharmaceutical products. Indeed, pharmaceutical research has produced a huge amount of active ingredients extremely effective, but sometimes difficult to administer because they are for example hydrophobic or too insoluble or having significant side effects. Current research is focused on improving the modes of administration of these new molecules to produce acceptable dosage forms according to the routes of administration, to explore new routes of administration, to target preferentially the foci of disease or to release drugs in a controlled manner to reduce the number of administrations or limit undesirable side effects.

 Current research therefore focuses in particular on the development of new systems for the release and stabilization of active ingredients.

 In the context of dispersed systems, there may be mentioned, for example, liposomes, generally consisting of a phospholipid sheet (Liposome: a practical approach, 1990, RRC New (Ed), LRL Press, Oxford University Press). Those skilled in the art can now manufacture uni or multi-lamellar liposomes, uni or multi-vesicular.

Liposomes can carry polar compounds indoors, they can also carry hydrophobic compounds in their phospholipidic layer as well as solubilize difficult compounds, thanks to the particularly detergent nature of the lipid sheet. These particles, however, have the disadvantage of being relatively unstable, difficult to manufacture in a controlled manner and having problems of leakage of the active ingredient.

 To overcome the problems of liposomes, other approaches have been proposed in order to increase the mechanical stability or the incorporation properties.

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 active ingredients. Lipobeads (Pin et al., FEBS Letters 1996,397, p.70-4), solid core liposomes (EP 277776 The University of Tennessee Research Corporation), BVSMTM (EP 687173 Biovector Therapeutics) are thus found. where the ionic matrix is inside the structure and allows to incorporate the assets by ionic interactions.

Regarding matrices or controlled release systems, there are: - amphiphilic hydrogels (WO 97/00275 GelSciences, Koller et al, STP
Pharma 1987,3, p.115-24): these gels are mainly used for hydrophobic or macromolecular active ingredients. Some of these gels have properties of state change depending on the temperature, which can be exploited at the release of assets (thermosensitivity).

- Cross-linked or non-ionic matrices, ionic (Irwing, Drug Development and
Industrial Pharmacy, 1987, 13, p.2047-66; WO 94/20078 Biovector
Therapeutics). Such matrices can be positively or negatively charged by grafting appropriate ionic charges and those skilled in the art are familiar with the various manufacturing processes. Those skilled in the art can manufacture particles of predetermined size from these matrices, for example by extrusion. These matrices can be used for the stabilization and release of polar compounds using ion exchange mechanisms. But these mechanisms are difficult to control.

 It was then sought to combine ionicity and hydrophobicity, in particular to be able to charge hydrophobic and charged active ingredients. In this context, block copolymers with charged hydrophobic and hydrophilic moieties (US Pat. These gels have been mainly studied because they can have changes of state depending on temperature, pH and ionic strength. It is clear that this mixture of hydrophobicity and ionicity is useful for the solubilization and stabilization of amphiphilic or insoluble compounds. However, a restriction of this system is that the hydrophobicity and ionicity distribution is not homogeneous throughout the gel and depends in particular on the size, shape and

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 the arrangement of the monomers being synthesized. We will therefore always have apolar parts and distinct ionic parts.

 Moreover, the physico-chemical properties of such matrices are determined by their synthesis and can not be modulated later. To obtain matrices with different properties of hydrophobicity or ionicity, different syntheses are necessary.

 The Applicant provides an original answer to this problem of developing new systems combining properties of hydrophobicity and ionicity.

 Thus, the Applicant has demonstrated new polymeric matrices which have very interesting properties of ionicity and hydrophobicity distributed homogeneously in said matrix. Furthermore, one of the advantages of the matrices according to the invention is that the hydrophobic and / or ionic character of the matrix can be modulated in a very simple manner, without the need to change many parameters.

 Thus, the invention relates to polymeric matrices, characterized in that they comprise a macromolecular, non-liquid hydrophilic matrix carrying a positive or negative ionic charge and in which is incorporated a lipid phase of charge opposite to that of the matrix.

 In a very interesting way, the polymer matrices according to the invention have an amphiphilic and ionic character, dispersed homogeneously in the matrix. This character can be modulated according to the quantity and nature of the lipids incorporated in the hydrophilic polymeric starting matrix.

 Preferably, the hydrophilic matrix consists of linear or branched polymer whose ionic charge is naturally or chemically grafted. The polymer may be natural or synthetic, homopolymer or copolymer (block, graft) or a random blend of polymers. Ionic polymers already containing hydrophobic parts (whether block copolymers or pendant chain copolymers) can also be used for the implementation of the invention, as long as the insertion of a lipid phase makes it possible to modulate the hydrophobicity of these polymers.

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 Preferably, the linear or branched polymer is a polyhydroxylated polymer, that is to say that each monomer has at least one free hydroxyl group.

 In a preferred embodiment of the invention, the matrix consists in particular of polysaccharides or oligosaccharides, but it is also possible to use other polymers, as the examples demonstrate. When a polysaccharide is used, it is preferably chosen from dextran, chitosan, starch, amylose or amylopectin or mixtures, cellulose, polygalactose, polymannose and their derivatives.

 The matrix of the invention may also be composed of natural or synthetic ionic polymers such as alginates or carbopol, known for their matrix-forming property spontaneously in the presence of multivalent ions.

 The polymeric matrix according to the invention comprises a hydrophilic matrix which can be crosslinked naturally or chemically.

 The methods for crosslinking polysaccharides or polyhydroxylated polymers are known to those skilled in the art and conventionally involve bifunctional reagents such as epichlorohydrin and bi-epoxides (leading to crosslinking of the di-ether type), phosphoric acid such as phosphorus oxychloride or trimetaphosphate (leading to phosphate diester type crosslinking) or dicarboxylic acids (leading to diester type crosslinking). This description is not limiting and other so-called conventional crosslinking agents can be used to obtain crosslinked hydrophilic matrices.

 The polymer matrix may be positively or negatively charged, by grafting ionic, cationic or anionic groups, to the monomers, preferably sugars, which make up the polymer. These groups may be chosen from quaternary ammonium functions or tertiary amines, phosphates, sulphates, carboxyethyls, succinates, etc. The charge grafting methods are well known to those skilled in the art.

 The lipid phase penetrating the polymers may consist of different types of natural or synthetic lipids, among which:

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 anionic lipids in general, in particular anionic phospholipids such as phosphatidyl glycerol or phosphatidyl serine (and in particular DPPG (diacyl phosphatidyl glycerol) and DPPS (diacyl phosphatidyl serine)), phosphatidic acids, anionic surfactants such as salts of long chain fatty acids, short or long chain fatty acids, cardiolipins, alkyl sulfates, sulphated glycerides, alkylpolyethoxy ether sulphates, alkylpolyethoxy ether carboxylates, alkyl sulphonates, mono or dialkyl phosphates - cationic compounds such as alkyl or dialkyl ammonium (in particular diacyl dimethyl ammonium, and acyltrimethylammonium), alkyl pyridiniums, cationic phospholipids and phosphonolipids, cationic surfactants, cationic lipids, in particular of the last generation such as DOGS, DOTAP , acylcarnitine, esters of long chain betaines.

 This list is not exhaustive. Examples of lipid molecules or ionic surfactants can be found in general galenic structures, for example: Puisieux and Seiller, Galenica 5. Surface agents and emulsions. 1989.

Tech. & Doc. Lavoisier.

 The lipids that can be used for the invention are water insoluble compounds, tending to be in micellar (detergent) form or in lamellar form (phospholipids). Penetration is induced by a supply of energy (temperature) to fluidize the lipids and allowing the ionic interaction to be effective. Once the lipids have penetrated, the drop in temperature allows the entrapment of lipids permanently.

 All charged lipids, however, are not suitable for the implementation of the invention. Indeed, lipids loaded too soluble will tend to leave the matrix when it is placed in an ionic medium by an ion exchange mechanism.

 The preferred lipids are those of the anionic phospholipid type or their cationic analogs, that is to say, charged groups derived by 2 or more fatty acid chains whose solubility in water is so low that it is considered that they are always in organized form.

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 With regard to the more conventional surfactant molecules or formed of an ionic group derived by a single hydrophobic chain, it is difficult to say where is the solubility boundary to obtain stable amphiphilic matrices. The Applicant has determined that sodium dodecyl sulfate surfactants are at the limit of this solubility.

 However, since complex mixtures of surfactants can be envisaged, the use of more hydrophilic surfactants may also be possible depending on the properties that are desired to give to the hydrophilic matrix.

 The Applicant has therefore shown that mixtures of lipids of charge opposite to the matrix are conceivable to modify the properties thereof and found that the penetration of lipids of opposite charge can also incorporate neutral lipids which would not have penetrated otherwise. The lipid phase may therefore also contain such lipids.

 Thus complex mixtures can be envisaged to very finely control the properties of the matrix. Indeed, in particular when it is desired to increase the overall hydrophobicity of the polymeric matrix according to the invention, without however greatly modifying the charge, it is possible to mix charged lipids and neutral lipids: cholesterol, neutral phospholipids, vitamins, etc. ..

 Surprisingly, the lipids incorporated in the hydrophilic polymer are organized in such a way that the hydrophobic zone is homogeneous and is stable in the matrix.

 Studies have shown that regardless of the amount and type of lipid incorporated, it is evenly distributed in the matrix. For example, in the case of anionic phospholipids which spontaneously form liposomes, it has been proved that these phospholipids are organized inside the matrix in an unknown form, but which has lipid bilayer characteristics and in particular the presence of a transition temperature.

 The Applicant has shown that the matrices compact more and more according to their lipid load. This compaction can only be explained by a hydrophobic interaction between the lipidic counter-ions and not by a charge neutralization phenomenon, since it has been shown that by neutralizing the charge with

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 counterions less lipid (short chain fatty acids), this compaction is no longer.

 The homogeneous distribution of the lipids loaded in the matrix and its compaction show that the resulting structure results from the competition between ionic interaction (which would tend to disperse the lipids in the matrix) and the hydrophobic interaction which tends to group the lipids. We can certainly speak of ionic and amphiphilic matrix.

 A second important consequence of this compaction is that vesicular amphiphilic matrices can be obtained thanks to this hydrophobic component.

 Indeed, it is possible to obtain amphiphilic matrices simply by using branched polymers of sufficiently large size. Thus, the Applicant has shown (Example 12) that by using cationized, very high molecular weight amylopectin (naturally branched polysaccharide) molecules (up to 100 million Daltons), vesicular amphiphilic matrices whose size can be obtained can be obtained. is about 100 to 250 nm. Indeed, the incorporation of the lipid phase of the opposite charge in this type of polymer causes a spontaneous contraction of the polymer molecules by hydrophobic interaction and the formation of nanometric amphiphilic matrices effective for the transport of active principles.

 The existence of such a unique structure makes it possible in particular to solve the problems posed during the encapsulation of active principles having properties rendering them little or not soluble in water, or which have amphiphilic characteristics.

 Thus, these very particular properties (a certain hydrophobicity, while keeping ionic charge characteristics) allow: - the suspension of very insoluble compounds, preferably those which have a charge opposite to the matrix (amphotericin, ketoconazole, antifungals in general, anti-cancer agents, etc.), - the suspension of highly hydrophobic compounds, preferably of charge opposite to the matrix (vitamins, trans-retinoic acid, hormones, antidepressants), but which can also be neutral

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 (especially apolar hormones such as estradiol, estradiol valerate, centrally acting prostaglandins), the stabilization of polar compounds of charge opposite to that of the matrix, which are incorporated by ionic interaction, and whose molecule may possess an apolar part, the stabilization of therapeutic peptides (in particular insulin, HgH, HPTH, antigens).

 The particular topology of the polymeric matrices according to the invention (consisting of an amphiphilic matrix) thus makes it possible to encapsulate and stabilize various active principles, possessing hydrophobic and / or polar properties, to convey them and to deliver them. The controlled and / or delayed delivery / release method will depend on the ionicity and hydrophobicity characteristics of the matrix, its size and / or shape. It is thus possible to formulate active ingredients which were unstable in previously developed systems, as well as to protect them from the surrounding medium in the case of, for example, oral administration.

Among these molecules, mention may be made, without this list being exhaustive: antibiotics and antivirals proteins, proteoglycans, peptides liposaccharides antidepressants hormones, antigens, insecticides and fungicides compounds acting on the cardiovascular system - compounds acting on the central nervous system - anticancer drugs - anti-malarics - anti-asthmatics - compounds with an action on the skin - nucleic acids (DNA, RNA, nucleotides, for example)
The form that will be decided to give subsequently to such a polymeric matrix will be defined according to the intended application. It can, in

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 in particular, be selected from the group comprising films, patch components, visible particles, microparticles, nanoparticles. Such derivatives of the polymeric matrix are also part of the invention.

 Whatever form it is desired to give to the matrix according to the invention, it should be noted that the lipids can be introduced into the interior of the initial matrix after this has been determined. Conversely, it is also possible to prepare the amphiphilic polymeric matrices according to the invention and to model them later.

 The Applicant has found that the lipid load capacity of these matrices is related to their ionic charge capacity. Thus, when it is desired to prepare dispersed nanoparticles, the lipid loading rate is limited to around 60-80% of neutralization of the charge. From this limit, precipitation phenomena are observed because the particles become too hydrophobic. They can then optionally be stabilized using conventional neutral emulsifiers, whether detergents, surfactants or phospholipids which are nonionic or zwitterionic.

 The different possibilities of shape of the matrix make it possible to envisage their use for the administration of molecules by: - intravenous - intramuscular route (including in deposit form) - transdermal - subcutaneous - oral route.

 These matrices according to the invention should maintain the known properties of mucoadhesion imparted by the ionic polymer, and it is even probable that these properties are increased by the hydrophobic part.

 Thus, these matrices are particularly effective for: - the release of molecules by the mucosal route (in particular nasal, vaginal, oral) - the transport of antigens for vaccine use (in particular by the mucosal route, especially via the nasal route) - the transport of oligonucleotides (DNA, RNA or the like) mucosally

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 the delivery of peptides, in particular mucosally.

 The compounds according to the invention can also be administered dermally, in particular as a patch.

 The polymeric matrices according to the invention can be sterilized. They can also be frozen.

 The invention also relates to a method for preparing a polymeric matrix, characterized in that: a) A hydrophilic polymeric matrix carrying a positive or negative overall charge is prepared. b) The solution of lipids of charge opposite to that of the matrix is prepared. c) A mixing of the two solutions is carried out with stirring, so that the lipid phase penetrates entirely into the polymer matrix or the core.

 Those skilled in the art will be able to optimize the conditions for the lipid phase to penetrate entirely into the polymer matrix or the nucleus (in particular the different proportions and the contact times). It may sometimes be necessary to heat the lipid phase to a sufficient temperature to fluidify and facilitate its entry into the polymeric phase. The temperature conditions depend on the type of lipid and the polymeric matrix used. However, in general, the temperatures useful for carrying out the process according to the invention will range from 20 to 95 ° C., preferably from 40 to 80 ° C.

 The advantages of these polymer matrices are in particular: a high chemical and thermal stability, due to the polymeric structure of the solid matrix, and interactions stabilizing the lipid phase inside thereof.

 the possibility of modulating and defining the overall charge and the hydrophobic character, by varying the polymers, their derivation rate and the lipids chosen for the preparation.

 - a great ease of synthesis and industrialization, with a reduced number of steps.

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 their ability to stabilize hydrophobic and / or polar compounds difficult to integrate into conventional vectors.

 the possibility of modifying the delivery of the active ingredient by modulating the overall charge and hydrophobicity of the particulate vector as well as its size and shape.

 - A muco-adhesion property for the use and delivery of the active ingredient, especially orally or mucosally.

 The active ingredients can be introduced into the polymer matrices at different stages of manufacture, depending on their charge and their hydrophobicity.

In particular, it is advantageous to produce the matrices and to charge them with the active principle thereafter, but it is also possible to introduce the active principle during the preparation of the lipid phase and / or the manufacture of the polymer with stirring. .

 The amphiphilic and ionic matrices according to the invention can therefore be used for the incorporation of active principles. It is remarkable that the small matrices (submicron), having incorporated an active ingredient can undergo a sterilizing filtration, including a sterilizing filtration under 0.2 m, without significant loss of material. Significant loss of equipment means a loss of not more than 15% of material, more preferably not more than 10%, most preferably not more than 5% material.

 The matrices according to the invention having incorporated an active principle possess extremely interesting properties. Indeed, the Applicant has highlighted the fact that the polymeric matrix-active ingredient complexes allow not only the solubilization of insoluble active ingredients, but also the protection and stabilization of said active ingredients. This makes it possible in particular to protect the active principles which are normally sensitive to hydrolysis, oxidation and other degradation reactions, which can be observed in vitro (and thus increase the stability and shelf life of the pharmaceutical or cosmetic compositions according to the invention), or in vivo (and thus increase the duration of action of the active ingredient used, and / or allow to reduce the doses administered).

 The matrices according to the present invention are also particularly suitable for the stabilization of active principles in suspensions in isotonic medium, in particular for an injectable application.

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 The matrices according to the invention, because of their chemical structure, are particularly well suited to oral or mucosal use, but can also be used for the administration of active ingredients parenterally or topically. Ophthalmic eye drops, ear preparations, mouthwashes, vaginal preparations, mouthwashes, pulmonary aerosols are considered.

 It is interesting to note that the matrices according to the invention make it possible to improve the effects related to these different types of administration (in particular the bioavailability and the stability of the active principles).

 As has been said previously, the fact of being able to modulate the particle size and / or the lipid composition also makes it possible to modulate the release rate of the active ingredient.

 Thus, the present invention is also directed to a method for modifying the release rate of an active ingredient, characterized in that it contains the steps of: a) preparing matrices according to claim 1, with variations in size and / or of lipid phase composition, with possible incorporation of said active principle, b) incorporation of said active principle in the matrices prepared in step a), if it was not incorporated in step a) c ) study of the modification of the release rate of the active ingredient according to the structure of the matrix.

 The study envisaged in step c) can be carried out in vitro, in particular in dissolution media, or in vivo, in appropriate animal models. Furthermore, it is possible to incorporate the active ingredient in the matrices directly during the preparation of said matrices or later.

 This method thus makes it possible to determine the matrix according to the invention to be chosen for the encapsulation of active principles, and the preparation of pharmaceutical or cosmetic compositions having predetermined active principle release properties. This is valid regardless of the chosen mode of administration.

 It should be noted that the solubilization of active ingredients in the form of matrices of very small size should have the important consequence of improving the

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 provision (rate and yield of absorption, bioavailability) of insoluble compounds after oral administration, or mucosal administration.

 In the context of compounds for which insolubility is the determining parameter, which leads to poor absorption, this should result in a noticeable improvement in intestinal absorption.

 It is thus possible to envisage using very small particles which will very quickly release the active ingredient in an available form, to prepare fast onset formulations. This is particularly sought in the case of analgesics and treatment of seizures.

 We can also consider the use of larger particles, releasing the active ingredient more slowly in the intestinal tract to obtain a prolonged prolonged release effect in the case where it is sought to avoid peak plasma levels while maintaining a constant plasma concentration. for the longest time possible.

 The subject of the invention is also a pharmaceutical composition, characterized in that it contains a polymeric matrix according to the invention in which an active ingredient is incorporated, and a pharmaceutically acceptable carrier for its administration. Those skilled in the art know how to choose acceptable carriers for pharmaceutical use. The matrices according to the invention are especially useful for therapeutic and vaccine applications. The use of matrices according to the invention as medicaments is also an object of the invention.

 The subject of the invention is also a cosmetological composition, characterized in that it contains a polymeric matrix as described above in which is incorporated an active principle with dermatological and / or cosmetic activity, and cosmetologically acceptable excipients.

 Finally, the food compositions comprising polymeric matrices according to the invention form part of the invention.

 The following examples serve to illustrate certain aspects of the invention, and should not be construed as limiting this invention.

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BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Fluorescence of the mixture of cationic particles / liposomes at room temperature. The penetration of lipids (fluorescent) into the matrix is studied by following the protocol of Example 5.

 2: Fluorescence of the mixture of cationic particles / Liposomes at 80 ° C. The penetration of lipids (fluorescents) into the matrix is studied by following the protocol of Example 5.

 Figure 3: Study of the hydration rate of Sephadex A-50 as a function of the level and the type of lipid counter-ion. The study is carried out following the protocol of Example 7. The symbols used are: diamond, CI 6; square, C8; triangle, DPPG.

 FIG. 4: Evolution of the fluorescence polarization of DPH as a function of temperature for amphiphilic matrices loaded with DPPG or palmitate.

 The study is carried out according to the protocol of Example 7. M.A .: amphiphilic.

The symbols used are: diamond, M.A. C16; square: M. A. DPPG.

 Figure 5: Evolution of the size of non crosslinked cationic amphiphilic matrices as a function of the lipid content and the polymer used. The study is carried out according to the protocol of Example 12. The symbols used are: diamond, cationized amylose; square, cationized amylopectin.

 Figure 6: Amphotericin B solubilization rate by uncrosslinked cationic amphiphilic matrices, in PBS. The study is carried out according to the protocol of Example 13. Symbols, AmB: B, +: cationized.

 Figure 7: Stability of submicron Amphotericin B formulations at room temperature expressed as the percentage of amphotericin filterable under 0.2 m. Symbols, diamond = water; square = 5% glucose; = PBS.

 Figure 8: Comparison of 2 formulations and control in terms of absorption of cyclosporin A in the contents of the bags. Symbols, rhombus = control; square = cyclo-1 preparation; triangle = cyclo-2 preparation.

 Figure 9: Comparison of 2 formulations and control in terms of tissue accumulation of cyclosporin A. Symbols, rhombus = control; = cyclo-1 preparation; triangle = cyclo-2 preparation.

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Figure 10: Pharmacokinetics of cyclosporin A blood obtained after oral administration of the formulations in the beagle. Symbols, rhombus = Neoral; square = cycle-1 preparation; circle = cyclo-2 preparation EXAMPLES Example 1: Preparation of liposomes DPPG
4 g of DPPG (5.58 mmol, Lipoid KG, Germany) are dispersed in 250 ml of an ethanol: water (88: 12) solution containing 3.4 g of choline acetate (21 mmol, Sigma, USA) . The solution is heated to 60 C until the complete dissolution of the phospholipids.

 12.5 ml of this solution of DPPG are injected into 87.5 ml of water at 80 ° C. with magnetic stirring, and the solution is then allowed to return to ambient temperature. A translucent suspension of DPGG liposomes at 2 g / l having a size around 100 nm, measured by light scattering in a submicron Coulter N4D particle analyzer (Coultronics, USA), is obtained.

 The solution is then stored at +4 C.

Example 2: cationic hydrophilic matrices of several micrometers based on polysaccharide
50 g of polysaccharide (amylopectin, Waxilys 200, Roquette Frères, France) are dispersed in 100 ml of water. When no more lumps, 25 ml of 10 M sodium hydroxide are added. The alkaline solution of polysaccharide is stirred until a translucent viscous solution is obtained.

 2.83 g of epichlorohydrin (ECH, 30 mmol, Sigma, USA) are then added and after one night of gentle stirring at room temperature the gellation of the polysaccharide is observed. 31.2 g of glycidyl trimethylammonium (GTMA, 200 mmol, Fluka, Switzerland) are added and the reaction is stirred for 8 hours at room temperature. At the end of the reaction, 1 liter of water is added and the pH of the solution is brought between 5 and 7 with acetic acid. The gel obtained is washed by carrying out several decantations in water and the suspension is pre-homogenized with an Ultra-Turrax model T-25 (IKA, Bioblock, France).

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 The suspension is then homogenized at a pressure of 50 bar using a Minilab H-80 homogenizer (Rannie, Denmark) to obtain fragments of 1 to 50 m which are visible under an optical microscope.

 The particle suspension is then centrifuged at 5000 g for 15 minutes (Sigma centrifuge 3k-20, Bioblock, France) and the pellet is recovered with osmosis water.

The suspension of particles is then dried by lyophilization and 20 g of cationized crosslinked polysaccharide particles having the following characteristics are obtained: Size (optical microscopy): 5-50 μm
Charge (Nitrogen Analysis): 1.75 mmol / g (quaternary ammonium groups per gram of particles).

Example 3 Preparation of submicron cationic hydrophilic matrices based on polysaccharide.

 50 g of a maltodextrin (Glucidex 6, Mw: 3400 polysaccharide hydrolyzate, Roquette Frères, France) are solubilized in 80 ml of water and then 20 ml of 10M sodium hydroxide are added. In the alkaline solution of polysaccharide maintained with stirring, 3.54 g of epichlorohydrin (38.3 mmol) are added. After a night of gentle stirring at room temperature, the solution gels.

 31.2 g of glycidyl trimethylammonium (200 mmol) are added to the gel and the reaction is left stirring for 8 hours. After the end of the reaction, 1 liter of water is added and the pH of the solution is brought between 5 and 7 with acetic acid. The suspension is pre-homogenized using an Ultra-Turrax T-25.

 The particle suspension is then homogenized (Minilab H-80) at a pressure ranging between 500 and 900 bar to obtain particles having a size of less than 100 nm.

 The particles are then purified by ultrafiltration on a 30 kDa membrane (Minissette system, Filtron, USA). The suspension of particles is then purified by microfiltration on a 0.2 m membrane (Spiral-Cap 0.2 m, Gelman, USA) and stored at 4 C.

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The characteristics of the suspension are:
Size (Coulter N4D): 60 nm (SD = 20 nm)
Charge (Elemental Analysis): 2 mmol / g
Potential Z (Malvem 3000 series, 15 mM pH 7 phosphate buffer): + 18 mV
Concentration: 17 g / 1 Example 4: Anionic hydrophilic submicron matrices based on polysaccharide
50 g of Glucidex 6 are solubilized in 200 ml of osmosis water and then 50 ml of 10 M sodium hydroxide are added. This solution is refrigerated at + 4 ° C. and maintained under stirring.

 23 g of phosphorus oxychloride (150 mmol, Prolabo, France) are added at the same time as 85.2 ml of 10M sodium hydroxide. The addition of these two products is carried out simultaneously and for 2 hours. At the end of the addition of reagents, the reaction is left stirring for 1 hour. In this case, the crosslinking and the functionalization are carried out at the same time because the phosphorus oxychloride has the property of crosslinking by phosphodiester functions and of grafting the filler by a phosphate monoester bond.

 1 liter of water is then added and the pH of the solution is brought between 5 and 7 with acetic acid.

 The solution is pre-homogenized with an ultra-turax and then homogenized between 500 and 900 bar to obtain particles smaller than 100 nm.

 The solution is purified by ultrafiltration on a membrane having a filtration threshold of 30 Kda and then microfiltered over 0.2 m.

A suspension of particles having the following characteristics is obtained:
Size (Coulter N4D): 50 nm (SD 20 nm) Total charge (total phosphorus analysis): 2 mmol / g
Crosslinking rate (phosphodiester functions deduced by titration): 0.2 mmol / g
Potential Z (15 mM phosphate buffer, pH 7): -21mV
Concentration: 20 mg / ml

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Example 5 Preparation of a Cationic Amphiphilic Matrix of Several Micrometers with anionic Phospholipids
10 ml of a suspension of 10 g / l of cationic hydrophilic matrices synthesized according to the method of Example 2 is mixed with a solution of liposomes DPPG at 2 g / l (10 ml) manufactured according to Example 1. The mixture is carried out at room temperature.

 In order to monitor the penetration of lipids into the cationic matrix, the DPPG suspension is pre-added with 50 g of a lipophilic fluorescent probe (5-HexaDecanoylAminoEosine). This hydrophobic and neutral probe follows the lipids in its distribution and is fluorescent only in the presence of lipids.

 A sample is observed under a fluorescence microscope (Nikon Eclipse T-300) and photographs are taken.

FIG. 1 shows a fluorescence preferentially on the surface of the particles indicating that, at ambient temperature, the lipid phase can not penetrate the polysaccharide network correctly. The interaction between anionic liposomes and cationic particles is evident but diffuse fluorescence can be observed in the background (excess liposomes) indicating saturation of the particle surface
The same sample is treated at 80 ° C. and then (FIG. 2) a much higher fluorescence penetration and a disappearance of fluorescence in the background are observed.

 It can also be observed that regardless of the size of the particles, the fluorescence seems to be distributed homogeneously in the volume of the particle.

Example 6: Submicronic cationic amphiphilic matrices with anionic phospholipids
24 ml of a 17 g / l solution (408 mg of particles) in a cationic hydrophilic matrix prepared according to Example 3 are stirred at 80 ° C.

 With the aid of a peristatic pump, 10.2 ml of an ethanolic solution of DPPG at 16 g / l (ethanol: water 88: 12, DPPG 163 mg) are added slowly into the cationic polysaccharide solution. After the addition of the entire DPPG, the solution

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 is stirred at 80 C for 30 min. The solution is purified by ultrafiltration on a membrane having a filtration threshold of 30 KDa.

 This results in a suspension of submicron amphiphilic matrices with a matrix: lipid ratio of 40% w / w. Taking into account the rate of charge of the matrix (2 mmol / g) and the molar weight of DPPG (722 Da), it can be concluded that the degree of neutralization of the cationic charge by the DPPG is 26%.

 This results in 34 ml of a suspension of submicron particles (100-150 nm measured by Coulter N4D) at 16.5 g / l. The Z potential of these amphiphilic matrices remains largely cationic (+15 mV measured in 15 mM phosphate buffer pH 7).

 Example 7: Different anionic lipids on submicron cationic matrices Characterization of the systems. Limitation of the type of lipids.

 In order to visualize the effect of the lipid phase on the physical properties of the hydrophilic matrix, we investigated the penetration of different anionic lipids on a commercial cationic hydrophilic matrix, Sephadex QAE A-50. These are cross-linked dextran particles, functionalized by quaternary ammonium functions and having a spherical shape and a size around 100 m, which makes them very useful for modeling.

 The effect of the combination of different types of lipids (DPPG, palmitic acid and octanoic acid) is studied on QAE Sephadex A-50 particles by measuring the modification of their swelling rate (hydration) for different amounts of lipids.

 On 300 mg of Sephadex at 10 g / l, different amounts of lipids are added (see Table I) and heated for 30 minutes at 80 ° C. The initial degree of hydration of Sephadex A-50 is 65 ml / g. The cationic charge rate is 3 mmol / g. For the same level of charge neutralization, the mass of lipids to be used is proportional to the molar mass of the lipid.

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Table I. Lipid mass ratio / hydrophilic gel for different lipids and different neutralization rates (for 1 g of Sephadex A-50).

<Tb>
<Tb>

<SEP><SEP><SEP> Neutralization <SEP><SEP><SEP><SEP> Load <10% <SEP> 33% <SEP> 65% <SEP> 100%
<tb> Sephadex
<tb> Mass <SEP> of <SEP> DPPG <SEP> (MW <SEP>: <SEP> 722) <SEP> 0.22 <SEP> g <SEP> - <SEP> 1.4 <SEP> g <SEP> 2.2 <SEP> g <SEP>
<tb> Mass <SEP> of <SEP> palmitic acid <SEP> (MW <SEP>: <SEP> 255) <SEP> 0.076 <SEP> g <SEP> 0.25 <SEP> g <SEP> 0 , 49 <SEP> g <SEP> 0.76 <SEP> g <SEP>
<tb> Mass <SEP> of <SEP> octanoic acid <SEP> (MW <SEP>: <SEP> 143) <SEP> 0.043 <SEP> g <SEP> 0.14 <SEP> g <SEP> 0 , 28 <SEP> g <SEQ> 0.43 <SEP> g
<Tb>

The swelling rate is measured for each type of lipid as a function of the rate of neutralization of the charge (Figure 3). It is observed that DPPG and palmitic acid behave in the same way, causing a sharp contraction of the gel due to the hydrophobic interactions between the fatty acid chains. On the other hand, octanoic acid is incapable of contracting the matrix, indicating too much solubility or mobility of fatty acid chains. In this case the lipid is probably in equilibrium between the matrix and the surrounding medium.

 To better understand these results we worked with submicron amphiphilic matrices prepared according to Example 7. Four types of matrices were prepared using four different anionic lipids (DPPG, sodium palmitate, sodium octanoate and sodium dodecyl sulfate). The four matrices were prepared at a neutralization rate of 20%. The four lipids were previously added with a lipophilic fluorescent probe (diphenyl hexatriene or DPH, Molecular Probes, the Netherlands).

 The four suspensions of hydrophilic matrices have similar characteristics (size around 100 nm and Potential Z around 15mV). We then measured the fluorescence emission (Perkin-Elmer Spectrofluorimeter LS 50B) in all four cases in water and physiological saline (PBS). Table II clearly shows that in the case of DPPG and palmitate the fluorescence level is similar in water and PBS, indicating good lipid stability within the matrix. For octaonate and dodecyl sulphate, the water fluorescence indicates a loading of the lipid matrix, but this fluorescence disappears completely in ionic medium, indicating a complete lipid leak.

<Desc / Clms Page number 21>

Table II Study of the fluorescence emission of different cationic amphiphilic matrices in water or in PBS as a function of the lipidic counterion.

<Tb>
<Tb>

Against <SEP> ion <SEP> Fluorescence <SEP> Fluorescence
<tb> in <SEP> water <SEP> (AU) <SEP> in <SEP> PBS <SEP> (UA)
<tb> DPPG <SEP> 425 <SEP> 395
<tb> Palmitate <SEP> 350 <SEP> 340
<tb> Octanoate <SEP> 250 <SEP> 0
<tb> Dodecyl <SEP> sulfate <SEP> 300 <SEP> 0
<Tb>

It can be concluded from this study that too soluble lipids or in any case surfactants with a solubility in water greater than that of sodium dodecyl sulfate alone can not form stable amphiphilic matrices.

 In both cases where the amphiphilic matrix seems stable (DPPG and palmitate) we have carried out a polarization study of the fluorescence of DPH as a function of temperature. In fact, the DPH makes it possible to determine the lipid transition temperature because its position in the lipid phases is different as a function of the fluidity state of the membrane. FIG. 4 represents the evolution of the fluorescence of the two types of matrices as a function of the temperature in an aqueous medium. The DPPG is observed to have a transition temperature around 45 ° C., indicating that the phospholipids are organized within the matrix in a lamellar form having the same cooperative characteristics as the liposomal forms. Palmitic acid, on the other hand, does not have a transition temperature, the progressive decrease in fluorescence is explained by the gradual increase in the fluidity of the lipid phase.

 Finally we carried out a study of the size of the particles obtained as a function of the degree of neutralization of submicron lipid matrices loaded with DPPG. We have observed that for neutralization rates higher than 60% of the load, we begin to observe aggregation phenomena due to too much hydrophobicity of the matrices. This phenomenon has also been observed for other lipids, and does not depend on the lipid load but on the degree of neutralization of the matrix, indicating that the ionic character must be maintained to prepare stable amphiphilic matrices in suspension.

<Desc / Clms Page number 22>

Example 8: Anionic Amphiphile Matrices of Sub-Micron Size with Cationic Lipids
10 ml of a solution of anionic matrices (3 g / l, 30 mg) prepared according to Example 5 are placed at 80 C. 200 l of an ethanolic solution of dioctadecyl dimethyl ammonium (30 g / l, 6 mg, Sigma, USA). After addition of the lipid, the suspension is incubated at 80 ° C. for 30 minutes and then allowed to cool to room temperature. The ethanol is then removed by ultrafiltration on a membrane of 30 Kda.

A suspension of 5 g / l anionic amphiphilic matrices having the following characteristics is obtained:
Size: 230 nm (SD 90 nm)
Lipid load 20% w / w
Potential Z (15mM phosphate buffer pH 7): - 14 mV Example 9: Solubilization and stabilization of Amphotericin B in cationic amphiphilic matrices.

 Amphotericin B is an extremely insoluble antifungal that has no charge at physiological pH. It can nevertheless be considered that it is an example of an insoluble compound of charge opposite to the matrix because the COOH function can be polarized by its interaction with the matrix.

 10 ml of a solution of amphiphilic vectors of Example 6 at 5 g / l (50 mg) is stirred in a water bath at 50 ° C. 500 μl of a solution of Amphotericin B are added to 20 g / l of dimethyl sulfoxide (DMSO) are added to the solution of amphiphilic vectors. The resulting suspension is stirred at 50 ° C. for 2 hours. The complete dissolution of Amphotericin B is observed.

 The suspension of particles obtained is filtered over 0.2 m and the level of association of Amphotericin B with the amphiphilic vectors is measured by spectroscopy (absorption at 405 nm). The association rate is greater than 90% in water.

<Desc / Clms Page number 23>

 1 ml of suspension of charged amphiphilic particles is then diluted with 9 ml of PBS buffer and the level of association is measured in the same way. The association rate in PBS remains above 80%.

 The stability of the Amphotericin B formulations thus obtained was monitored over time in water and in various isotonic media such as 5% glucose or isotonic phosphate buffer (PBS). Figure 7 shows excellent stability of the size of the formulations either at 4 C or at room temperature for several months.

Example 10 Incorporation and Stabilization of a Very Hydrophobic Compound in Cationic Amphiphilic Submicron Matrixes

 Estradiol valerate is a highly hydrophobic estradiol derivative.

 8.9 mg of estradiol valerate are solubilized in 5 ml of an ethanolic solution of DPPG at 16 g / l prepared according to Example 1.

 According to the method described in Example 6, 2.8 ml of DPPG-estradiol solution (45 mg DPPG, 5 mg of estradiol valerate) are injected into 20 ml of a suspension of cationic hydrophilic matrices prepared according to the example 3 (3.5 g / l, 70 mg).

After dialysis at 37 ° C. against water, a translucent suspension of amphiphilic matrices loaded with estradiol valerate having the following characteristics is obtained:
Size: 100-150 nm
Potential Z (15 mM phosphate buffer pH 7) = + 1 1 mV
Water association rate (HPLC):> 90%
Combination rate in PBS (HPLC):> 80% Example 11 Incorporation of a neutral lipid with the charged lipid in the amphiphilic matrix.

 An ethanolic DPPG / cholesterol solution: 70/30 (w / w) at 20 g / l is prepared starting from an ethanolic solution of DPPG at 16 g / l prepared according to Example 1.

 This ethanolic solution of DPPG / cholesterol (1 ml, 20 mg) is injected under heat into 10 ml of a solution of submicron cationic vectors prepared

<Desc / Clms Page number 24>

 according to Example 3 to 3 g / 1 (30 mg) which is maintained at 80 C. The lipid content is 65% w / w (45% DPPG, 20% Cholesterol) relative to the vectors.

The suspension is kept for 30 minutes at 80 ° C. and is then allowed to return to ambient temperature. A suspension of submicronic 5 g / l cationic amphiphilic matrices having the following characteristics is obtained:
Size: 90 nm (SD 30 nm)
Cholesterol association rate: 100%
Potential Z (15 mM phosphate buffer, pH 7): + 15mV.

 Example 12: Submicronic cationic amphiphilic matrices from non-crosslinked cationized natural polymers.

 In this example, the natural polymers studied are amylose (linear type polysaccharide) and amylopectin (branched type polysaccharide).

Both polymers have very high molar masses.

 Both polysaccharides are cationized according to the method described in Example 2 without adding epichlorohydrin. They are treated with GTMA under the same conditions so as to obtain cationized polymers having a loading rate of 1.7 mmol / g in quaternary ammonium functions.

 The non-crosslinked cationic amphiphilic matrices are then prepared according to the same methodology as Example 7. The size of these matrices is studied as a function of the level of anionic phospholipids (DPPG, FIG. 5).

 We first observe a compaction of the cationic polymer molecules, probably due to the hydrophobic interaction between the fatty acid chains. This compaction spontaneously produces amphiphilic matrices of size close to 200 nm. In the case of amylopectin it is observed that these matrices are formed from 5% neutralization (6% DPPG w / w)) and remain in the same size range up to 60% (80% DPPG w / w ) of neutralization, a value from which aggregation strongly increases. In the case of amylose, the aggregation is progressive from 5% neutralization. It seems that branched polymers form better structured amphiphilic matrices probably due to a naturally more globular form of the molecule.

<Desc / Clms Page number 25>

 The amphiphilic matrices obtained from the cationized amylopectin therefore have a size around 200 nm and are stable when placed in a saline medium (PBS).

 Example 13: Amphotericin B in uncrosslinked amphiphilic matrices

 Incorporation is carried out according to the method of Example 8 from uncrosslinked amphiphilic matrices of Example 12. The amphiphilic matrices used were carried out at a DPPG level of 40% w / w.

 The solubilization rate of Amphotericin B (AmB) is studied according to the type of non-crosslinked amphiphilic matrix. The degree of solubilization of Amphotericin B in water shows little difference with the conventional amphiphilic matrices already described, but the stability of the association in PBS shows strong differences (FIG. 6).

 It is observed that for low Amphotericin concentrations, both types of matrices are capable of stabilizing amphotericin as well as crosslinked matrices. On the other hand, for higher levels, only the matrices obtained from amylopectin are able to maintain amphotericin in suspension, at a rate slightly lower than that of the crosslinked matrices.

EXAMPLE 14 Preparation of submicronic anionic amphiphilic matrices loaded with halofantrine

 Halofantrine is a known antimalarial drug effective against most resistant forms of Plasmodium, but with significant intestinal absorption problems related to its lack of solubility.

 An aqueous suspension of submicron anionic matrices according to Example 4 at 15 g / l (100 ml, 1.5 g) is prepared which is maintained at 70 ° C. 10 ml of an ethanolic solution of dipalmitoyl phosphatidyl choline are added at 20 ° C. g / l (DPPC, 200 mg) and 2 ml of an ethanolic solution of cetyl trimethyl ammonium bromide at 40 g / l (CTAB, 80 mg).

 7.5 ml of an ethanolic halofantrine solution at 40 g / l (300 mg) are then added.

<Desc / Clms Page number 26>

 The whole is incubated for 15 minutes at 60 ° C. and then brought to room temperature.

Ethanol is removed by diafiltration against water on a 200 ml Amicon 8200 system equipped with a 30 Kd cutoff YM-30 membrane. The final volume is adjusted to 150 ml.

A suspension of submicronic amphiphilic matrices loaded with halofantrine and containing cationic lipids and neutral lipids is thus obtained.

<Tb>
<Tb>

Submicron <SEP> Particles <SEP>: <SEP> 10 <SEP> mg / ml <SEP> (72 <SEP>%) <SEP>
<tb> DPPC <SEP>: <SEP> 1.33 <SEP> mg / ml <SEP> (9.5 <SEP>%) <SEP>
<tb> CTAB <SEP>: <SEP> 0.53 <SEP> mg / ml <SEP> (3.8 <SEP>%) <SEP>
<tb> Halofantrine <SEP>: <SEP> 2 <SEP> mg / ml <SEP> (14.4%)
<tb> Yield <SEP> of incorporation <SEP>: <SEP> 100 <SEP>% <SEP> (HPLC)
<tb> Halofantrine <SEP> Filterable <SEP> under <SEP> 0.2 <SEP> m <SEP>: <SEP>><SEP> 95 <SEP>% <SEP> (HPLC)
<tb> Size <SEP>: <SEP> 120 <SEP> nm <SEP> (Coulter <SEP> N4D)
<Tb>
EXAMPLE 15 Preparation of submicronic (100 nm) and micronic (5-50 m) cationic amphiphilic matrices loaded with Cyclosporin A.

* Cyclo Formulation 1
1 g of dipalmitoyl phosphatidyl glycerol (DPPG) is dispersed with 4 ml of a solution of Cyclosporin A in ethanol at 100 g / l (400 mg). 8.5 ml of a 20 g / l sodium palmitate solution (170 mg) and 90 ml of water with stirring are added. The solution is maintained at 60 C.

 An aqueous suspension of submicron cationic particles prepared according to the method described in Example 3 is prepared at 20 g / l (2 g, 100 ml). This solution is maintained at 60 C.

 The solution of lipids and cyclosporin is added to the solution of submicron particles with stirring at 60 ° C. and the formulation is then brought back to ambient temperature.

This gives 200 ml of a suspension of submicron amphiphilic particles whose characteristics are as follows:

<Tb>
<tb> Submicron Particles <SEP>: <SEP> 10 <SEP> g / 1 <SEP> (56 <SEP>%) <SEP>
<tb> DPPG <SEP>: <SEP> 5 <SEP> g / 1 <SEP> (28 <SEP>%) <SEP>
<tb> Ac. <SEP> Palmitic <SEP>: <SEP> 0.85 <SEP> g / l <SEP> (4.8 <SEP>%) <SEP>
<Tb>

<Desc / Clms Page number 27>

<Tb>
<tb> Cyclosporine <SEP> A <SEP>: <SEP> 2 <SEP> g / 1 <SEP> (11.2%)
<tb> Size <SEP>: <SEP> 100 <SEP> nm <SEP>(<SEP> Coulter <SEP> N4D)
<tb> Yield <SEP> incorporation <SEP> Cyclo <SEP> A <SEP>: <SEP> 100 <SEP>% <SEP> (HPLC)
<tb> Cyclo <SEP> A <SEP> Filterable <SEP> under <SEP> 0.2 <SEP> m <SEP>: <SEP>><SEP> 95 <SEP>% <SEP> (HPLC)
<Tb>
* Cyclo Formulation 2
The same lipid solution is added to a suspension of cationic particles of 5-50 μm prepared according to Example 2 containing 20 g / l of particles (100 ml, 2 g) and which is maintained at 60 ° C.

 The results are of the same type as those presented previously, except the size which remains between 5 - 50 m and the filterability under 0.2 m which is no longer feasible.

Example 16: Intestinal passage on an evoked sac model in the rat.

Protocol
The various cyclosporine formulations were prepared according to Example 15, replacing palmitic acid with sodium dodecyl sulphate (SDS). For the control formulation, cyclosporine was incorporated into SDS micelles. The formulations studied Cyclo-1 and Cyclo-2, correspond to different sizes of nanoparticles, respectively 100 nm and 5-50 m. These formulations are aqueous solutions of a cyclosporin / [3H] -cyclosporine mixture at a concentration of 10 mM. By mixing 1 ml of these solutions with 9 ml of TC199 medium (95% O2, 5% CO2), 10 ml of a 1 mM cyclosporine solution was obtained.

 Male rats (Sprague-Dawley) of about 350 grams, deprived of food for 24 hours, are sacrificed by cervical stretching. The intestine is removed in its entirety and cleaned in 0.9% NaCl solution at room temperature. It is then immersed in the oxygenated TC 199 medium at 37 ° C. (10 ml of concentrated TC 199, 5 ml of 44 mg / ml NaHCO3, 1 ml of glutamine at 1 mg / ml, 20% H 2 O, 100 ml). The pH of the medium after oxygenation is 7.4. By means of a glass rod, the intestine is eversed and filled with oxygenated medium. From the jejunum, small bags, 2 to 3 centimeters, are made using silk threads. Each of them is placed in an Erlenmeyer flask containing 10 ml of incubation medium at 37 ° C. with shaking (60 cycles per minute). The control bags are dipped in the middle and left immediately. To the

<Desc / Clms Page number 28>

 At the end of the desired incubation time, the bags are rinsed by rapid passage in 4 0. 9% NaCl baths. The bags are weighed after drying on absorbent paper. Their content is recovered to be counted. The empty bag obtained is again wiped and weighed.

 The difference in weight makes it possible to estimate the volume of its contents (density of the medium: 1,008 g / ml at 37 ° C.). The empty bags are dissolved in 1M sodium hydroxide at 37 ° C. with stirring for 3 hours.

 The protein assay is performed on each bag dissolved by spectrophotometry at 750 nm (Perkin Elmer spectrophotometer, Lambda 3) according to the method of Lowry (Peterson, 1986) and using as standard bovine serum albumin.

 For the counting of the radioactivity, volumes of 400 l taken from the contents of the bags and 500 l taken from the incubation medium are filled to 1 ml with distilled water. 800 l volumes of tissues dissociated with sodium hydroxide are removed and neutralized with 200 l of 5N HCl. The determination of the radioactivity in the samples supplemented with 5 ml of scintillant (Ready Safe, Beckman) is carried out on a Beckman LS 1801 counter.

Results
The results of the absorption in the contents of the bags are given in FIG. 8. The results of the tissue accumulation are given in FIG. 9.

 There is a passage of cyclosporine through the intestinal mucosa in the model of the bag of evacuated rat intestine. Cyclosporin formulated with 0.1 m size particles (1-cyclo-1 formulation) was best absorbed in the contents of the bags. There is a negative effect of the particle size on the absorption, the formulation made from vectors of 5-50um, did not cause a significant increase in the passage of cyclosporine. The vectors of size 0.1 m even showed a higher efficiency compared to those of 5-50 m at time 75 minutes.

 With respect to tissue adsorption, particles of 5-50 m size resulted in an increase in absorption over control at 30 and 45 minutes. During the experiment, these particles formed aggregates with the mucus secreted by the intestinal sacs and were contiguous to the intestinal mucosa.

<Desc / Clms Page number 29>

This observation goes in the direction of a significant muco-adhesion of larger particles.

 These results make it possible to conclude that: the vectors of the invention promote the passage of cyclosporine in the model of the bag of intestines of rats.

 - A smaller size of these vectors is a positive factor for the release of the active ingredient through the eversed bags.

 the increase in the tissue concentration of the active principle is probably due to a strong mucoadhesion of the particles carrying the cyclosporin.

Example 17: Bioavailability of Cyclosporine in Beagle Dog protocol
4 beagle dogs weighing approximately 10 kg are treated in cross-over according to the design presented in the table below:
Table III: Pharmacokinetic Study in Beagle Dog

<Tb>
<tb> N
<tb> Period <SEP> 5056 <SEP> 6899 <SEP> 0667 <SEP> 5828
<tb> 1 <SEP> A <SEP> B / <SEP> D
<tb> 2 <SEP> D <SEP> A <SEP> B /
<tb> 3 <SEP> / <SEP> D <SEP> A <SEP> B
<tb> 4 <SEP> B <SEP> E <SEP> D <SEP> E
<Tb>

At different times, after oral administration of the formulations described below, 3 ml of blood are withdrawn and the blood concentration is measured by cyclosporin A by LC-MS using cyclosporin U as internal standard.

 Formulations A: cyclosporin A fluid in water in combination with ABV type vectors as described in Example 15 (cyclo-2 formulation, 1.2 mg / ml Cyclosporine-A and 9.5 mg / ml ABV ref KX)

<Desc / Clms Page number 30>

 Formulations B: cyclosporin A fluid in water in combination with ABV type vectors described in Example 15 (Cyclo 1 formulation, 1.22 mg / ml Cyclo-A and 9.5 mg / ml ABV ref KX) .

The reference formulations are as follows:
Formulation D: Neoral capsules
Formulation E: Sandimmun capsules.

 Both of these products are available from Novartis Pharma, and are described in the Vidal dictionary.

Results
Figure 10 gives the profile of blood pharmacokinetics of cycloporin A after oral administration of different formulations. compared to the reference (Neoral). Tables IV to VI summarize the pharmacokinetic parameters derived from these curves.

The analysis of the results shows: a bioequivalence of the formulations made from ABV vectors of 100 nm (formulation B) compared to the Neoral reference. Thus, the results of formulation B and Neoral in terms of bioavailability are perfectly equivalent (99% Cmax and 100% AUC 0-48H Neoral). In addition, formulation B has a coefficient of variation on the
Cmax lower than that of Neoral @ (20 vs 39%), suggesting that the absorption obtained with this formulation is less dependent on inter-individual factors.

 - Expectedly Sandimmun is less well absorbed than Neoral (70% Cmax and 66% AUC 0-48H). Formulation B is in this case better absorbed than this one (Table V and IV).

a negative effect of the increase in particle size (formulation B versus formulation A). Formulation B (100nm liquid) is the best absorbed which is visualized on both AUC and Cmax (Table IV and
V) - this same formulation has early adsorption compared to Neoral '(formulation D) and Sandimmun (formulation E) (Table VI).

<Desc / Clms Page number 31>

 Thus, the results obtained make it possible to demonstrate the action of the formulations made from ABV type vectors on the bioavailability of a relatively insoluble active ingredient in water (cyclosporin A) and suggests that these formulations could make it possible to act on the speed of passage of the active ingredient into the bloodstream.

Tables IV to VI: Pharmacokinetic parameters of the various formulations tested after oral administration at the beagle

<Tb>
<tb> AUC <SEP> 0-48H <SEP> (ng.h / ml)
<tb> Animal <SEP> Form <SEP> A <SEP> Form <SEP> B <SEP> Form <SEP> D <SEP> Form <SEP> E
<tb> 5056 <SEP> 3610.84 <SEP> 4945.39 <SEP> 5077.35 <SE> NS <SEP>
<tb> 6899 <SEP> 2250. <SEP> 75 <SEP> 3842. <SEP> 86 <SEP> 3933.83 <SEP> 3812.17
<tb> 0667 <SEP> 1647.28 <SEP> 2289.33 <SEP> 2402.64 <SEP> @ <SEP> ND
<tb> 5828 <SEP> ND <SEP> 5499. <SEP> 62 <SEP> 5227.39 <SEP> 2224.93
<tb> Average <SEP> 2502.95 <SEQ> 4144.30 <SEQ> 4160. <SEP> 30 <SEP> 3018. <SEP> 55
<tb> Deviation <SEP> 1005. <SEP> 78 <SEP> 1415.44 <SEP> 1306.43 <SEP> 1122. <SEP> 34
<tb> standard <SEP> (SD)
<tb> Coefficient <SEP> of <SEP> 40% <SEP> 34% <SEP> 31% <SEP> 37%
<tb> variation <SEP> (CV)
<tb>% <SEP> vs <SEP> Neoral # <SEP> 66% <SEP> 100% <SEP> 100% <SEP> 66%
<tb> Table <SEP> IV
<tb> Cmax <SEP> (ng / ml)
<tb> Animal <SEP> Form <SEP> A <SEP> Form <SEP> B <SEP> Form <SEP> D <SEP> Form <SEP> E
<tb> 5056 <SEP> 499.60 <SEP> 757.10 <SEP> 700. <SEP> 20 <SEP> NA
<tb> 6899 <SEP> 273. <SEP> 00 <SEP> 714.00 <SEP> 1049.30 <SE> 793.40 <SEP>
<tb> 0667 <SEP> 310. <SEP> 90 <SEP> 484. <SEP> 20 <SEP> 413.60 <SEP> NA
<tb> 5828 <SEP> NA <SEP> 796. <SEP> 60 <SEP> 604.50 <SEP> 369.50
<tb> Average <SEP> 361. <SEP> 17 <SEP> 687.98 <SEP> 691.90 <SE> 581. <SEP> 45
<tb> SD <SEP> 121. <SEP> 38 <SEP> 139. <SEP> 98 <SEP> 266. <SEP> 39 <SEP> 299.74
<tb> CV <SEP> 34% <SEP> 20% <SEP> 39% <SEP> 52%
<tb>% <SEP> vs <SEP> Neoral # <SEP> 50% <SEP> 99% <SEP> 100% <SEP> 70%
<tb> Table <SEP> V <SEP>
<Tb>

<Desc / Clms Page number 32>

<Tb>
<tb> Tmax <SEP> (Hours)
<tb> Animal <SEP> Form <SEP> A <SEP> Form <SEP> B <SEP> Form <SEP> D <SEP> Form <SEP> E <SEP>
<tb> 5056 <SEP> 2 <SEP> 1 <SEP> 1.5 <SEP> NA
<tb> 6899 <SEP> 1 <SEP> 0.5 <SEP> 1 <SEP> 1
<tb> 0667 <SEP> 1 <SEP> 1 <SEP> 2 <SEP> NA
<tb> 5828 <SEP> NA <SEP> 1.5 <SEP> 2 <SEP> 1.5
<tb> Average <SEP> 1.3 <SEP> 1.0 <SEP> 1.6 <SEP> 1.3
<tb> Table <SEP> VI
<Tb>

Claims (10)

 1. Polymeric matrix characterized in that it comprises a macromolecular hydrophilic matrix carrying a positive or negative ionic charge and in which is incorporated a lipid phase of opposite sign to that of the matrix. 2. Polymeric matrix according to claim 1, characterized in that, in the matrix and / or in the lipid phase, is incorporated an active ingredient. 3. Use of a polymeric matrix according to one of claims 1 or 2, for the preparation of a polymer matrix complex - active ingredient intended to be administered orally. 4. Use of a polymeric matrix according to one of claims 1 or 2, for the preparation of a polymeric matrix-active ingredient complex intended to be administered mucosally, parenterally or topically. 5. Use according to claim 4, characterized in that said complex is in a form selected from the group consisting of ophthalmic eye drops, ear preparations, mouthwashes, vaginal preparations, mouthwashes, and pulmonary aerosols. 6. Use of a polymeric matrix according to one of claims 1 or 2, for the preparation of a polymeric matrix-active ingredient complex, allowing the protection and / or stabilization of said active ingredient sensitive to hydrolysis reactions, oxidation and other degradation reactions. 7. Use of a matrix according to claim 2 for the preparation of a polymeric matrix-active ingredient complex, characterized in that said active ingredient is an antigen, said complex being administered by injection or mucosally. 8. Process for modifying the release rate of an active ingredient, characterized in that it contains the steps of: a) preparing matrices according to claim 1, with variations in size and / or composition of lipid phase , with possible incorporation of said active ingredient, <Desc / Clms Page number 34>  b) incorporation of said active ingredient in the matrices prepared in step a), if it has not been incorporated in step a) c) study of the change in the release rate of the active ingredient as a function of the structure of the matrix. 9. Use of a polymeric matrix according to one of claims 1 or 2, for the preparation of a polymeric matrix-active ingredient complex intended to improve the bioavailability of said active ingredient relative to the active ingredient not formulated, whatever the route of administration. 10. Use of a polymeric matrix according to one of claims 1 or 2, for the preparation of a polymeric matrix-active ingredient complex intended to be administered orally or mucosally and to improve the absorption of said active principle.