EP0999827A1

EP0999827A1 - Novel liposome vectors of active principles

Info

Publication number: EP0999827A1
Application number: EP98930820A
Authority: EP
Inventors: Jean-Pierre Salles
Original assignee: Lipogel
Current assignee: Lipogel
Priority date: 1997-06-11
Filing date: 1998-06-11
Publication date: 2000-05-17
Also published as: HUP0002069A2; RU2203649C2; CN1264297A; AU738809C; FR2764508B1; US20020146447A1; FR2764508A1; HUP0002069A3; PL337757A1; AU738809B2; JP2002511078A; AU8112398A; KR20010013665A; US6656497B2; NZ501795A; BR9810258A; WO1998056352A1

Abstract

The invention concerns liposome vectors, in powder form, of active principles, and more particularly active principles sensitive to digestive and/or plasmatic degradation, such as proteins, and their application as medicine. Said liposome vectors of active principles consist of a powder composition essentially constituted of unilamellar liposomes comprising an external lipid phase consisting of class 4 lipids (phospholipids), optionally associated with class 2 substances, class 3 substances and/or class 5 substances and an internal aqueous nucleus consisting of a mixture M of at least two different non-polymerisable gelling agents (G1 and G2) whereof the gel-sol phase transition is not less than 37 °C, G1 being selected among gelatines and carrageenans and G2 being selected among carrageenans with properties different from the carrageenans selected for G1, and celluloses, which liposomes have a diameter ranging between 20 nm and 1 mm, preferably between 20 nm and 500 nm; said composition having the form of particulate units with an average particle diameter between 10 mm and 1000 mm, formed by one or several of said liposomes, enclosed in a sheath selected in the group consisting of a dehydrated thermoreversible aqueous gel identical to said internal nucleus aqueous gel, dextrins or a mixture thereof, such that they contain, on an average, 10?16 to 1018¿ liposomes per gram of powder; and at least an active principle contained, as the case may be, either in the gelled internal nucleus or in the external lipid phase.

Description

NOVEL LIPOSOMAL VECTORS OF ACTIVE INGREDIENTS.

The present invention relates to stable liposomal vectors, in powder form, of active ingredients, and more particularly of active ingredients sensitive to digestive and / or plasma degradation, such as proteins, as well as to their application as a medicament. .

To protect such fragile active principles, numerous vectors have been proposed; among these, mention should be made of liposomes, which have been considered as a vector of choice.

The first studies of oral liposome administration have not been conclusive (DESHMUKH DS. Et al., Life Sciences, 1981, 28, 239-242). The results obtained showed that the liposomes of formulation: diether-phosphatidylcholine (non-digestible analogues of PC) / cholesterol-7: 1 allowed gastrointestinal protection of the encapsulated peptide, but did not allow its passage through the barrier. intestinal. Several reasons can be put forward to explain this non-passage: size of the liposomes too large and not calibrated, poor stability of the structure or leakage of the encapsulated compound towards the extra-liposomal medium.

Recently, the team of Robert Greenwood (Dr g Dev. And lnd. Pharm.,

1993, 19, 11, 1303-1315), from Campbell University in the USA, succeeded in showing that the duodenal intubation of insulin-carrying liposomes caused a hypoglycaemic effect greater than that obtained after a duodenal intubation of a free insulin solution.

Numerous attempts have been made to obtain liposomes having good capacities for transporting the active ingredients, in particular as regards the action on the percentage of capture of the active ingredient, the stability of the liposomes and the bioavailability of the active ingredient. We can cite, for example, as an indication:

- SB Kulkarni et al. (J. Microencapsulation, 1995, 12, 3, 229-246) which review the factors involved in the microencapsulation of drugs in liposomes: size of the liposome, type of liposome, load on the surface of the liposome, rigidity bilayer, addition of encapsulation adjuvants. It emerges from this evaluation that MLVs (multilamellar vesicles) containing several bilayers and with a diameter between 100 nm and 20 mm are desirable for the encapsulation of hydrophobic drugs interacting with bilayers, while LUVs (large unilamellar vesicles) containing a single bilayer and with a size between 100 and 1,000 nm are considered to be the most suitable for encapsulating hydrophilic drugs.

- I. De Miguel et al., (Biochimica and Biophysica Acta, 1995, 1237, 49-48), which propose nanoparticles composed of an internal nucleus formed of crosslinked polysaccharides, grafted to their external part by fatty acids and surrounded a layer of phospholipids; - P.S. Uster et al., (FEBS Letters, 1996, 386, 243-246) which propose to insert phospholipids modified by poly (ethylene glycol) in preformed liposomes to improve bioavailability.

Series of experiments relating to the administration of peptides by the oral route have been carried out and use either different methods of liposomal encapsulation, or modification of the lipid active principle by grafting of lipophilic functions. In all cases, the aim is to transform the lipid active principle into a "prodrug"; this prodrug has the property of resisting gastrointestinal transit, namely resistance to gastric pH, physiological detergents (bile salts), proteases (intestinal exo- and endo-peptidases) and metabolism by the intestinal flora. For example, the bridging in position 2 of a 1,3-diglyceride on a pentapeptide made it possible to confer these qualities on the drug thus modified.

However, these different liposomes of the prior art do not allow both good stability, an acceptable yield of encapsulation of the active ingredient and a significantly improved bioavailability per os of said active ingredient, without modifying the active ingredient, which thus retains all of its functions and properties. Bioavailability means the fraction of the dose that reaches the systemic circulation in pharmacologically active form and the speed with which it reaches it. J.C. Hauton, described liposomes with gelled internal nucleus

(lipogelosomes ^® ) in suspension in an aqueous medium containing gelling substances. He has, in particular, developed a process making it possible to manufacture such liposomes (European patent 0 393 049), which differs from conventional liposomes in that the encapsulated aqueous phase is in semi-solid gel form and not in liquid form, which prevents liposome fusions in collisions. Such lipogelosomes ^® are produced only from natural substances, which minimizes the risks of intolerance. In particular, in European Patent 0393049, these lipogelosomes ^® consist of a bilayer interfacial phase in the case of unilamellar lipogelosomes or a plurality of interfacial phases bilayer superimposed concentrically, in the case of multilamellar lipogelosomes ^® and by a gelled encapsulated internal aqueous polar phase, in which the polymerizable or non-polymerizable gelled substance is selected from polysaccharides, polypeptides or polyacrylamides; for example, the non-polymerizable gelable substance is selected from gelatin, agarose, or carrageenans and the polymerizable gelable substance is selected from polyacrylamide gels. These lipogelosomes ^® have a significantly increased stability, compared to the liposomes of the prior art, in particular due to the absence of interparticle fusion during collisions.

However, they have the disadvantage of being in the form of a dispersion of liposomes in the liquid phase, not suitable for the preparation of solid formulations, for easy storage and administration.

Consequently, the Applicant has set itself the goal of providing a new vector, which effectively makes it possible to obtain both a sufficient encapsulation yield and a significantly improved bioavailability per os of said active principle, compared with the liposomes of l 'Prior art, while exhibiting great stability both in storage and in vivo. Such vectors are suitable for oral administration; the aqueous solution is also suitable for other routes of administration: transdermal, pulmonary, nasal, genital, intravenous, subcutaneous or ocular, for example, depending on the excipient selected.

Said vectors are characterized in that they consist of: - a pulverulent composition which essentially consists of unilamellar liposomes comprising an external lipid phase consisting of lipid class 4 (phospholipids), possibly associated with class 2 substances (long chain triglycerides, cholesterol esters), class 3 substances (cholesterol, long ionized long chain fatty acids) and / or class substances 5 (bile salts, fusidic acid derivatives) and an internal aqueous nucleus forming a thermoreversible aqueous gel irradiating to the external lipid phase, which internal aqueous nucleus consists essentially of a mixture M of at least two gelling agents non-polymerizable Gl and G2, different and whose gel-sol phase transition point is greater than or equal to 37 ° C, Gl being a gelling agent selected from gelatins and carrageenans, such as kappa-carrageenans and G2 being selected from carrageenans having properties different from carrageenans selected for Gl, such as iota-carrageenans and celluloses, such as hydroxypropylm ethylcellulose, which liposomes have a diameter between 20 nm and 1 mm, preferably between 20 nm and 500 nm and which is in the form of particulate units having an average diameter between 10 mm and 1000 mm, formed by one or more of said liposomes, surrounded by a gangue selected from the group consisting of a dehydrated thermoreversible aqueous gel identical to the aqueous gel of said internal nucleus, dextrins or a mixture thereof, so that it comprises, in medium, 10 ¹⁶ to 10 ' ⁸ liposomes / g of powder and

- At least one active ingredient included, depending on the case, either in the internal gelled nucleus, or in the external lipid phase of said composition.

Surprisingly, such vectors make it possible to overcome the drawbacks associated with conventional liposomes. Indeed, they allow:

- to increase the stability of the liposomes, due to the absence of interparticle fusion during collisions, - to increase the bioavailability of the active principle (protection in the gastrointestinal tract and passage through the intestinal barrier); in particular, in rats, the time for passage of the vectors according to the invention (LGS) through the intestinal barrier from their administration by the oral route can be between 2 and 4 hours: either 1 hour of gastric emptying and 1 to 3 hours of passage from the intestinal lumen to the systemic circulation; thus an active ingredient with little or no cell internalization capacity can effectively be incorporated in a differentiated intestinal epithelial cell, when it is encapsulated in a vector (LGS) according to the invention, without modification of the activity or of the composition of the active principle.

- to decrease the toxicity of the encapsulated active ingredients and - to cause less leakage of the encapsulated products, due to the lower molecular mobility in the gelled encapsulated aqueous phase.

Unexpectedly, by selecting the gelling agents, it is possible to obtain liposomes (SUV or small unilamellar vesicles), suitable for use in a dry form (powder) and having particularly advantageous properties as a vector for active principles; indeed, surprisingly, the bioavailability by bone of said active ingredients, preferably active ingredients, sensitive to digestive degradation, poorly absorbed or very toxic, is significantly increased when they are encapsulated or associated with the vector according to the present invention. In addition, such vectors in pulverulent form maintain the entire integrity of the liposomes which they contain and which remain stable over time, both in pulverulent form and when they are resuspended, due to the maintenance of the integrity. constitutive lipids (no degradation product) and maintaining the integrity of the characteristics of the gelling agents, in particular of the Gl and G2 mixture (viscosity, gel and breaking strength, molecular weights).

The advantage of using lipogelosomes ^® (LGS) in this context is to benefit from a stabilized liposomal form (JC Hauton et al, Eur. J. Surg., 1994, suppl. 574, 117-119) in view administration of active ingredients orally. The LGS manufacturing method makes it possible to obtain on average gaseous hydrophilic phase encapsulation rates close to 10%. This percentage varies, in particular as a function of the molecular weight of the active principle, and is calculated according to the ratio of quantity of encapsulated active principle / quantity of active principle used. For example, at least 5% encapsulation is observed for a molecule of 500 Da and at least 50% encapsulation for a molecule of at least 20 kDa. With regard to peptides for example, 10 to 50% of encapsulation is observed, while generally for all the active ingredients, the percentage of encapsulation varies from 5 to 80%, depending on the case.

The gelling agents G1 and G2 differ in particular with regard to viscosity, molecular mass and the gel-sol transition point (that is to say the melting temperature). For Gl gelling agents, this temperature is less than or equal to 45 ° C, while it is greater than or equal to 45 ° C for G2 gelling agents.

The mixture M of at least two gelling agents Gl and G2 as defined above has texturometric characteristics (gel strength and breaking strength) which are particularly advantageous from the point of view of the stability of the liposomes obtained and the bioavailability of the principle. encapsulated asset. Thus, preferably, the mixture M of at least two gelling agents Gl and G2 has, at 5 ° C, relaxation characteristics of between 70 and 100%, preferably 81-89%, and a breaking force included between 1000 and 1600 g, preferably 1109-1503 g. According to another advantageous embodiment of said composition, said internal aqueous nucleus of the liposomes further comprises at least one stabilizing agent of osidic nature, and / or at least one agent for regulating the osmolarity of the medium and / or the at least one surfactant, such as a bile salt and / or a nonionic surfactant. Advantageously, said vectors comprise in% (m / m):

25 to 75% of class 4 lipids, 5 to 45% of gelling agents, 0 to 70% of stabilizing agent of osidic nature, 0 to 15% of agent for regulating the osmolarity of the medium, 0 to 20 % of surfactants and 0 to 15% of dextrins, preferably 8 to 12%; this formulation does not include the active ingredients. According to another advantageous embodiment of said pulverulent composition according to the invention, said aqueous internal core comprises 70 to 95% of Gl gelling agent and 5 to 30% of G2 gelling agent.

According to another advantageous embodiment of said pulverulent composition according to the invention, the stabilizing agent of an osidic nature is sucrose, trehalose or any other protective agent. The present invention also relates to a process for preparing the powder vectors according to the invention, in which the external gangue of the particulate units comprises a fraction of thermoreversible aqueous gel, characterized in that it comprises the following steps: (1) preparation of a dispersion of gelled internal core liposomes

(lipogelosomes ^® ) in the aqueous phase by (a) preparation of a solution of at least one suitable gelling agent, in particular a mixture M of gelling agents Gl and G2, by dissolving said gelling agents, with slow stirring, at a temperature higher than the gel-sol phase transition temperature of said gelling agents in an aqueous solution at pH compatible with the active principle to be encapsulated, (b) incorporation of the active principle in the solution obtained in (a), (c) incorporation of lipids in the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum and formation of an emulsion and (d) obtaining said dispersion of liposomes with internal gelled nucleus (lipo- gélosomes ^® ) in an aqueous phase containing said gelling agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum and

(2) obtaining the pulverulent product by suitable drying of the dispersion obtained.

According to an advantageous mode of implementation of said method, the drying is carried out by atomization, coacervation, thin layer or granulation.

The present invention also relates to a process for preparing the powder vectors according to the invention, in which the external gangue of the particulate units comprises a fraction of thermoreversible aqueous gel and / or a dextrin, characterized in that it comprises the following stages : (1) preparation of a dispersion of gelled internal core liposomes

(lipogelosomes ^® ) in the aqueous phase by (a) preparation of a solution of at least one suitable gelling agent, in particular a mixture M of gelling agents Gl and G2, by dissolving said gelling agents, with slow stirring, at a temperature higher than the gel-sol phase transition temperature of said gelling agents in an aqueous solution at pH compatible with the active principle to be encapsulated, (b) incorporation of the active principle in the solution obtained in (a), (c) incorporation of lipids in the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum and formation of an emulsion and (d) obtaining said dispersion of liposomes with gelled internal nucleus (lipogelosomes ^® ) in an aqueous external phase containing said gelling agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum,

(2) elimination of at least part of the aqueous liquid phase containing said gelling agents, in which the liposomes are dispersed,

(3) addition of at least one suitable dextrin and

(4) obtaining the pulverulent product by spray drying of the product obtained in (3).

According to an advantageous embodiment of said method, step (2) of eliminating at least part of the aqueous liquid phase containing said gelling agents is carried out by dilution and / or by filtration.

In accordance with the preparation methods according to the invention, the aqueous solution of step (a) further comprises an agent for regulating the osmolarity of the medium (0.9% NaCl, for example) and / or a stabilizing agent of osidic nature and / or a surfactant, preferably class 5 substances (bile salts).

As a variant, the active principle is added in the external lipid phase, before its incorporation into the mixture obtained in (a).

For example, calcitonin is incorporated at pH 5, AZT at pH 7.5 and doxorubicin at pH 3.

Surprisingly, such methods make it possible to obtain a vector in powder form liposome internal stable gelled core (lipogelosomes ^®) in a single step including a maturation phase (as defined in ripening) of the constituents in aqueous phase, at slow speed, then a dispersion phase (formation of lipogelosomes ^® ) at rapid speed, comprise a stage during which a stable dispersion of lipogelosomes ^® in liquid phase, of homogeneous morphology, which is capable of being obtained, is obtained subjected to the drying step; such a dispersion of gelled internal core liposomes has, in fact, the following morphology: . vesicular structure with a diameter between 20 nm and 500 nm, preferably between 20 and 80 nm,

. microscopic observations in negative staining, cryofracture, cryotransmission and atomic force: vesicles or assemblies of vesicles of appearance characteristic of phospholipid bilayers; negative staining makes it possible to observe the more or less marked presence of mixture M of gelling agents coating the external phospholipid layer, and

. polydispersity of liposomes with gelled internal phase of between 10 and 55%, preferably between 10 and 30%. Such a process has the advantage of being reproducible and perfectly adaptable on an industrial scale.

It also has the advantage of being less cumbersome to implement than the processes of the prior art in which a step of sonication, of extrusion or of elimination of detergents is necessary, as described in Patent 0 393 049.

According to an advantageous embodiment of said methods, step (c) is carried out, preferably, at a shear rate lower than

200 s ¹ ; in general, the shearing speed is given by the following ratio: speed of the stirring module / spacing between the internal wall of the reactor and the distal end of the stirring blade (also called "air gap").

The present invention also relates to a pharmaceutical composition, characterized in that it comprises a pulverulent liposomal vector of active principle as defined above and at least one pharmaceutically acceptable vehicle. According to an advantageous embodiment of said composition, it is in solid form (capsule, tablet, powder to be dissolved in water).

According to another embodiment of said composition, it further comprises an activator of cAMP.

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the process which is the subject of the present invention as well as in the appended drawings, in which:

- Figure 1 represents the variations of the calcemia as a function of time (-D- = free calcitonin; -M- = vector LGS-calcitonin according to the invention); FIG. 2 represents the difference in AUC between the calcemia obtained with free calcitonin and that obtained after administration, orally, of the LGS-calcitonin vectors according to the invention;

FIG. 3 represents the variations of calciuria as a function of time (-M- = vector LGS calcitonin 500 kDa, -D- = free calcitonin, -D- = vector LGS calcitonin 300 kDa);

- Figure 4 shows the evaluation of phosphatemia as a function of time (-D- = free calcitonin; -____- = LGS-calcitonin vector according to the invention);

- Figure 5 shows the difference in AUC between the phosphatemia obtained with free calcitonin and that obtained after administration, orally, LGS-calcitonin vectors according to the invention;

- Figure 6 shows the variations of phosphaturia as a function of time (-_____- = LGS calcitonin vector (construction (P A- vector) of molecular weight at least greater than 500 kDa, which is equivalent to lipogelosomes ^® encapsulating calcitonin diameter at least greater than 40 nm), -D - free calcitonin, calcitonin LGS -D- = vector (construct (P A-vector) of molecular weight at least greater than 300 kDa, which is equivalent to lipogelosomes ^® encapsulating the calcitonin with a diameter at least greater than 20 nm);

- Figure 7 shows the variations of the SGOT (IU / 1) as a function of time (-D- = free calcitonin; - - = vector LGS-calcitonin according to the invention); - Figure 8 shows the variations of SGPT (IU / 1) as a function of time (-D- = free calcitonin; - - = LGS-calcitonin vector according to the invention);

- Figure 9 shows the AUC differences in SGPT levels between the groups treated with free calcitonin and those treated with an LGS-calcitonin vector according to the invention. It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1 Texturometry measurements of the mixture of the gelling agents Gl and G2 a) Material and methods

The measurements are carried out on a TA-XT2i device from the company Rhéo. The study concerns the behavior of gels made up of a mixture of gelatin, iota and carrageenan kappa during rupture and relaxation tests. . Concentration of samples:

Gelatin / iota / kappa carrageenan mixture (80 / 17.5 / 2.5) at 7.5% w / V, in ₅ mM Na ₂ HPO ₄ medium and 0.9 or 2% NaCl.

. Preparation of a solution of gelling agents:

The sodium chloride is dissolved in a mixer fitted with a turbine and a sun gear and containing the purified water (15 minutes at 10 revolutions / minute), the mixer is raised to temperature up to 75 ° C. (stirring at 10 revolutions / minute for 45 minutes), the gelling agents (gelatin, iota carrageenans, kappa carrageenans) are added to the mixer at 75 ° C., the stirring turbine is started at 1500 revolutions / minute; the duration of the dissolution step is approximately 30 minutes; dissolution is complete when the solution is clear and does not contain suspended particles.

. Sample preparation:

For the relaxation test, 45 ml of gel are poured hot into a flat-bottomed Petri dish 92 ± 2 mm in external diameter. For the rupture test, 30 ml of gel are poured hot into a flat bottom crystallizer of 50 ± 2 mm in external diameter. The gel is obtained by cooling to a temperature less than or equal to 37 ° C. The maturation time of the gels, which corresponds to maximum hydration of the gels, is 2.5 days at study temperature and at rest.

Operating conditions: For the relaxation test, a compressive force is applied to the gel for a determined time. The mobile used is an aluminum cylinder of 25 mm in diameter with a pre-speed of 1.0 mm / s, a speed of 0.5 mm / s and a post-speed of 10.0 mm / s. The movement of the mobile is 1.0 mm for 30 seconds.

For the rupture test, the mobile used is a 10 mm diameter ebonite cylinder with a pre-speed, a speed and a post-speed of 1.0 mm / s. The movement of the mobile is 12 mm. b) results of a study at 5 ° C, with a NaCl level of 0.9% Relaxation (%) minimum value: 81 ± 2.2 maximum value: 89 ± 0.8

Breaking force (g) minimum value: 1109 ± 25 maximum value: 1503 ± 35 c) results as a function of temperature and different NaCl levels

The operating conditions are identical to those described in a), except for the displacement of the mobile used in the relaxation test (displacement of 20% of the total thickness of the gel). Relaxation (%) at 5 ° C

0.9% NaCl: 89 ± 0.8 2% NaCl: 90 ± 0.2 at 25 ° C

0.9% NaCl: 32 ± 3.9 2% NaC1: 38 + 4.4 at 37 ° C

0.9% NaCl: 36 ± 3.7 2% NaCl: 40 ± 4.9 Breaking force (g) at 5 ° C 0.9% NaCl: 1413 ± 66 NaC12%: 1114 ± 143 at 25 ° C

0.9% NaCl: 211 ± 2.7 NaC2%: 173 ± 1.5 at 37 ° C

0.9% NaCl: 25.7 ± 2.4 2% NaCl: 45.7 ± 3.9 EXAMPLE 2: Process for the preparation of a powder vector according to the invention containing calcitonin

1) Preparation of a dispersion of gelled internal phase liposomes (lipogelosomes®):

- Constituents:

Soy lecithins 11.915 kg (7.943%) Gelatin B150 7.149 kg (4.766%)

Iota carrageenan 1,565 kg (1,043%)

Carrageenan kappa 0.222 kg (0.148%)

Sucrose 8.936 kg (5.957%)

Sodium chloride 1,073 kg (0.715%) Purified water 119.15 kg (79.43%)

TOTAL MATERIAL 150.01 kg (100%). a) Preparation of a dispersion of liposomes a mixture of:

- gelatin B 150 7.149 kg - iota carrageenans 1.565 kg

- 0.222 kg carrageenan kappa

- sucrose 8.936 kg -NaCl 1.073 kg

- Na ^{+ -} chenodeoxycholate 1.131 kg - purified water 118.00 kg, (qs l50 kg) is premixed in a mixer at a speed of 10 revolutions / minute, the planetary of which rotates at the speed of 1500 revolutions / minute for 1.5 hours, under vacuum. b) Incorporation of calcitonin:

Using concentrated acetic acid (6N), the pH of the mixture is lowered by successive additions, until a stable pH of 4.5 is reached. Next, 4.075 g of salmon calcitonin (Bachem California), the specific activity of which is 7017 IU / mg, is added. c) Incorporation of the phospholipids in the solution obtained in a):

The soy lecithins (11.915 kg) are added to the premix, in a mixer at the speed of 10 revolutions / minute, in which the planet gear rotates at the speed of 1500 revolutions / minute, for 5 hours, under vacuum (-. Formation of 'an emulsion).

Final dispersion by increasing the planetary (25 rpm) and turbine (2,500 rpm) agitation speeds sufficient time to obtain a polydispersity of less than 40%.

A dispersion of lipogelosomes ^® in the aqueous phase is obtained. Microscopic observations in negative staining, cryofracture, cryotransmission and atomic force microscopy: vesicles or assemblies of vesicles of characteristic appearance of phospholipid bilayers; negative staining makes it possible to observe the more or less marked presence of external gelling agent according to the manufacturing and / or separation process chosen. d) tangential filtration

A volume of the dispersion of lipogelosomes ^®, obtained in the preceding steps was diluted in 20 volumes of 0.9% NaCl, hot, stirring. The diluent (0.9% NaCl) will be added with 8.25 × 10 ⁻⁴ % of chenodeoxycholate, according to the presence of this surfactant, in the preceding dispersion. The non-encapsulated phase is removed by tangential ultrafiltration continuous hot. Ultrafiltration is performed on a membrane with selective porosity of 300 or 500 kDa, depending on the particle size desired, lipogelosomes ^® . The product obtained is a lipogelosomes suspension ^®, encapsulating at least 17% of salmon calcitonin, wherein the diameters of the liposomes varies from 20 nm to 500 nm, when the suspension is ultrafiltered through a 300 kDa and of 40 nm to 500 nm, when the suspension is ultrafiltered to 500 kDA. 2) Drying of the dispersion obtained:

The dispersion of the lipogelosomes ^® in aqueous phase obtained is transferred to a vacuum dryer (50-100 mbar) for approximately 4 hours. A fairly homogeneous, straw-yellow, very clear powder is obtained, containing grains with a diameter of between 0.1 mm and 1 mm. At the level of electron microscopy observations, there has been a retraction of the lipid vesicles on themselves, due to dehydration. In addition, we note that, while in the liquid state, the LGS are often aggregated inside a homogeneous gelled gangue in an environment of numerous isolated vesicular structures, the drying step transforms this gelatinous gangue into filaments of gelling agent dry on the surface of the aggregates, but also on the surface of isolated vesicular structures.

Alternatively, the drying is performed as follows: the dispersion of lipogelosomes ^® in aqueous phase is distributed directly onto a rotating dryer cylinders (cylinder temperature: 120-150 ° C, rotation speed of 3-6 revolutions / min.). The “shavings” obtained are then ground and calibrated on an appropriate grid. This gives a powder lipogelosomes ^® (also referred to hereinafter LGS), having the characteristics defined above.

Drying can be optimized by adding a loading excipient, for example maltodextrin or β-cyclodextrins EXAMPLE 3: Comparative effects of free or encapsulated calcitonin in vectors obtained according to Example 2, after oral administration in the rat.

The effects of a preparation according to Example 2, on calcemia, calciuria, phosphatemia and phosphaturia, are analyzed compared to the oral administration of calcitonin in free form. The pharmacokinetics obtained for the two forms of calcitonin administered are also compared. Other parameters are also analyzed: transaminases (SGOT and SGPT) and blood sugar.

It is important to note that the effect of calcitonin in rats or in normocalcemic humans is difficult to demonstrate, and that the responses to this hormone are much clearer when pathological subjects (hypercalcemic) are treated.

Experimental protocol . Preparation of LGS-Calcitonin. See example 2. Animals and pharmacological treatment.

Animals

10 groups of 10 Wistar Ico rats (IOPS AF / Han, IFFA CREDO) or 100 rats in total, 6 weeks old and weighing between 160 and 180 g, were formed.

The weight of the animals is measured at the start of the experiment in order to ensure, at this parameter, a homogeneous distribution of the rats in each of the groups.

The 6 experimental groups are previously fed for 7 days on a sterile “AO4” basic diet (UAR = Rational Food Factory (Diets supplier)). The rats are fasted and under glucose ad libitum 24 hours before the administration of the experimental doses.

The weight of the animals is checked before administration of the experimental doses.

Experimental design The groups A, B, C, D, E, F, G, H, I, J are carried out as follows:

* group A: 10 control rats whose plasma and urine are collected at time 0.

* group B: 10 rats are intubated and 1.8 ml of LGS-Cal-500 kDa suspension (approximate calcitonin concentration: 54 IU / rat or 330 IU / kg) are administered to each individual. Plasma and urine are collected at time 45 min. * group C: 10 rats are intubated and 1.8 ml of LGS-Ca suspension / 500 kDa (approximate calcitonin concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 90 min. * group D: 10 rats are intubated and 1.8 ml of LGS suspension-

Cal-500 kDa (approximate calcitonin concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 180 min.

* group E: 10 rats are intubated and 1.8 ml of LGS-Cal-500 kDa suspension (approximate calcitonin concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 300 min.

* group F: 10 rats are intubated and 1.8 ml of free calcitonin suspension (concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 45 min.

* group G: 10 rats are intubated and 1.8 ml of free calcitonin suspension (concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 90 min.

* group H: 10 rats are intubated and 1.8 ml of free calcitonin suspension (concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 180 min.

* group I: 10 rats are intubated and 1.8 ml of free calcitonin suspension (concentration: 54 IU / rat or 330 IU / Kg) are administered to each individual. Plasma and urine are collected at time 300 min. * group J: 10 rats are intubated and 1.8 ml of LGS suspension

Cal-300 kDa (approximate calcitonin concentration: 36 IU / rat or 228 IU / Kg) are administered to each individual. Plasma and urine are collected at time 90 min.

Anesthesia: anesthesia is performed using Rompun ^®

(Xylazine 2%; 10 mg / kg) / Imalgene ^® (Ketamine 10%; 60 mg / Kg) by intraperitoneal injection according to the chronology indicated in the experimental scheme. Specimens

The blood samples from the abdominal aorta by catheterization under anesthesia are taken at times 0 for group A; time 45 min. for groups B and F; time 90 min. for groups C, G and J; time 180 min. for groups D and H; time 300 min. for groups E and I. The bladder is also cannulated and the urine collected at the same timing as that used for blood samples.

The total plasma will be obtained after separation of the blood samples by centrifugation at 3000 rpm, 15 minutes, in tubes containing 3.8% of EDTA (non-protein anticoagulant).

Analyzes

On each plasma sample, the calcemia, the phosphatemia, the transaminases will be assayed by colorimetry.

Statistical data processing The results of the measurements are expressed as an average ± SEM of the ten rats in each group. The data will be compared using statistical tests appropriate to this type of experimental protocol (parameter studies and pharmacokinetics). The statistical test chosen is the ANOVA test or analysis of variance, the meanings of the differences are determined by the Fisher test and by the Scheffe test which is more discriminating.

Two methods of expression of the results were used: the graphic representation of the means of 10 values relating to the parameter considered as well as the comparative analysis of the AUC (areas under the curve). This mode of expression makes it possible to appreciate the differences in amplitudes of the responses obtained.

The results obtained are represented using the pharmacokinetic technique: variation of the rate of the parameter considered as a function of time. It is not, in this case, the search for a dose effect. Results. Pharmacokinetics of the effect of free or encapsulated calcitonin in the form of LGS-Calc, on calcemia. Figure 1 shows the variations in calcemia as a function of time. The assays used to determine the calcium concentrations were carried out by the colorimetric method recorded in the Pharmacopoeia. The basal values of calcemia (at time 0) are very well correlated with previous data. Each point represents the average of 10 values, or 9 groups of 10 independent rats. The means are expressed ± SEM. The results are compared by analysis of variance (ANOVA), for unpaired values. Significant differences are symbolized by **. This symbol corresponds to a significance in the very discriminating Scheffe test. There is a transient decrease in serum calcium after oral administration of free calcitonin. It is explained by the fact that during a massive administration of peptide type calcitonin, a small percentage crosses the intestinal barrier (1%), without being denatured. Here 330 IU were administered which corresponds to a lymphatic passage of 3.3 IU (passage from the intestinal lumen to the plasma, via the lymphatic ducts). However, the IV route of calcitonin begins at 0.9 IU. It is therefore normal to observe this effect of free calcitonin. The hypocalcemia observed after oral administration of calcitonin decreases over time to return to normal after 90 min.

With regard to LGS-Calc, the same effect at 45 min is observed, but this hypocalcemic effect is twice as strong at 180 min. This fact indicates that the LGS formulation, with equivalent calcitonin concentration, is more effective in terms of pharmacological effect than free calcitonin. We can attribute this two-phase phenomenon to the activity of calcitonin linked to the outer layer of LGS (first action), the second effect could be due to the calcitonin contained inside LGS. There is therefore a delayed effect doubled by an increase in PA activity by a factor of 2.

FIG. 2 represents the difference in AUC between the calcemia obtained with free calcitonin and that obtained after oral administration of LGS-Calc. The difference observed is highly significant on the Scheffe test. The AUC corresponds to a total of all the values obtained during the experiment; these values are integrated and then compared. AUC corresponds to the area under the curve variations in calcemia over time. The lower this AUC, the greater the hypocalcemic effect (since the curve then approaches the abscissa axis).

. Pharmacokinetics of the effect of free or encapsulated calcitonin in the form of LGS-Calc on calciuria.

The restrictions mentioned concerning the plasma results obtained by atomic absorption are confirmed by the analysis of the values obtained on the urine of rats treated with free or encapsulated calcitonin. Indeed, figure 3 corroborates the values of figure 1, since hypocalcemia is always followed by an increase in calciuria.

. Pharmacokinetics of the effect of free or encapsulated calcitonin in the form of LGS-Calc, on phosphatemia. (figure 4)

LGS-Calc and free calcitonin induce hypophosphatemia (colorimetric assay) which continues only in the case of groups treated with LGS-Calc. The results are significant on the Fisher test. The comparison represented in FIG. 5, of the respective AUCs very clearly confirms the pharmacokinetic data.

The difference between the two AUCs is significant on the Scheffe test. . Pharmacokinetics of the effect of free or encapsulated calcitonin in the form of LGS-Calc on phosphaturia. (figure 6)

The assays on the urine samples were carried out by atomic absorption as in the case of calciuria (see above).

These results are less significant than in the case of calciuria. It is therefore difficult to conclude. Nevertheless, it seems that at time 180 min., The effect of LGS-Calc tends to be greater than that of the non-encapsulated drug.

. Toxicological aspect of the study.

Thanks to the samples taken, it was possible to carry out the assays for transaminases during the administration of the two active ingredients.

Analysis of SGOT levels over time shows a tendency to hypotoxicity (Figure 7) of the encapsulated form of calcitonin compared to the free form. This difference is very significant in the Fisher test at time 300 min. Nevertheless, the Comparisons of AUCs do not show significant differences in terms of changes in SGOT rates over time.

This tendency to moderate the increase in transaminases ("hypotoxicity") is confirmed by the analysis of SGPT levels over time (Figure 8).

These data show a strong hypotoxicity of the encapsulated form of calcitonin compared to the free form. Figure 9 shows the AUC differences in SGPT levels between the groups treated with free calcitonin and those treated with encapsulated calcitonin. The difference between the two areas is significant on the Scheffe test.

This effect can in particular be used in the context of administration of highly toxic active ingredients, in order to reduce the hepatotoxic impact of such substances.

Conclusion - The encapsulation of calcitonin in the LGS form potentiates the crossing of the intestinal barrier.

- The compared effect of administration by the oral route shows a real potential of the LGS form, all the more since it is now possible to stabilize this structure in powder form. - The biphasic hypocalcemic effect of LGS-Calc can be explained by the distribution of calcitonin on the surface and in the heart of LGS.

- This experiment makes it possible to highlight the hypotoxicity of the LGS-Calc form compared to the free form, which seems more toxic.

- The data demonstrating the superiority of the LGS-Calc vs free Calcitonin form were acquired using the dosage method recommended in the pharmacopoeia.

- The two peaks of hypocalcemia caused after oral administration of the two forms of PA were specified: 45 and 180 min after administration. - The "delay" effect of LGS-Calc could be due to a gradual release in the intestine of microspheres from a mother matrix: the LGS-Calc concentrates, which gradually penetrate the intestinal barrier. EXAMPLE 4 Increase in the bioavailability of the principles encapsulated in the lipogelosome and derivative forms; comparison between liposomes and lipogelosomes ^® .

1. Comparison of resistance or stability of the lipogelo-some ^® (LGS) and liposome (LS) classic forms.

LGS make it possible to produce dosage forms (powder) impossible to produce with conventional liposomal forms; alone, LGS are resistant to physiological conditions: pH, temperature, intestinal motility, enzymes, which gives them the ability to be administered by oral or pulmonary route, while LS are destructured, when administered by such tracks. a) Resistance to pH and intestinal bile salts: Series of LGS and LS incubations, 1 hour at 37 ° C in the presence of bile salt (taurodeoxycholate) with detergent power, and therefore destructive to vesicles lipids, are carried out. The results show that for a concentration of 0.25 mM in bile salt, the LGS are 3 times more resistant than the LS. The resistance of the structures is analyzed by laser granulometry (variation of the counting rate of the particles, indicated by the variation of the diffraction of a laser, in Khz).

The comparison of the structure (observed in laser granulometry) of LGS and LS after 1 hour of incubation at variable pH, shows that the LGS are stable at pH = 2.5 to 9 while the LS are mainly intact, only at pH = 6.3.

LGS are more resistant than LS to pH and detergent concentrations found in the stomach; this makes it possible to deduce that the LS are degraded in the stomach, while the LGS resist longer. b) Resistance to the serum medium, to temperature and to agitation: Series of LGS and LS incubations were carried out during

24 hours at 37 ° C with stirring. The lipid phases of LGS and LS, strictly identical in composition, have been labeled in the same way with an isotope ( ¹⁴ -C). The products resulting from the degradation of two types of structures: LS or LGS have been analyzed over time. It appears from the results that the lipid components of LS are released more easily than the lipid components of LGS. These results show that the LGS form is more stable than the form

LS. c) Comparison of the leakage of active ingredients encapsulated from LGS and LS:

A small active ingredient (PA) (500 Da) was encapsulated in LGS and in LS, in the same amount. Subsequently the two preparations were stirred at 37 ° C in a serum medium, and the release of the encapsulated PA was measured. The amount of PA released from LS is 60% greater than the amount of PA released from LGS (1.6 PA unit for LS; 1.01 PA unit for LGS). Thanks to this significantly superior stability of LGS, solid dosage forms are achievable and administration by the oral route is possible, whereas they were not possible with liposomes of conventional formulation.

2. Comparison of the bioavailability of the lipogelosome ^® and conventional liposome forms, in a cellular model.

We analyzed the differences in cellular internalization of a marker or a PA when these molecules are encapsulated in forms LGS (lipogélosome ^®) or SS forms (liposome). a) Comparison of cellular internalizations of liposomes and lipogelosomes®:

LS and LGS were labeled using radioactive probes or fluorescent probes, and, after an incubation period in a medium in the presence of human macrophages (strain THP1) in culture at 37 ° C, internalization comparison of the two types of structures (LS and LGS) was analyzed at the end of the incubation, the duration of the incubations being identical. The analysis of internalizations was carried out by various analytical methods. A. fluorescence microscopy

The images show an internalization of LS and LGS, but in the case of LS the signal distribution is homogeneous while the distribution of the LGS signal is localized in punctiform intracellular structures. This result shows that the LGS are degraded less quickly at the intracellular level than the LS structures (where the signal diffuses more quickly in the cell).

Thus, an effect of slow diffusion of the active ingredients encapsulated in LGS appears, while it does not exist for LS. B. radiolabelling

The count at the intracellular level of radiolabelled LGS and radiolabelled LS shows that LGS are 2.5 times more internalized than LS under the same experimental conditions.

LGS are internalized in greater quantity than LS: this fact shows that the cellular bioavailability of LGS is greater than that of LS, this fact is due to the difference between the two liposomal structures: the presence of a particular thermoreversible gel , in the internal phase of the LGS, irradiating to the surface of the particle, gives the LGS a preferential cell uptake property. C. flow cytometry These experiments are based on the comparative cellular endocytosis of LGS and LS labeled with a fluorescent probe. After incubation, the cells are harvested and then passed to a flow cytometer, which quantifies the fluorescent signal in each cell. The spectra obtained show that cells incubated with LGS emit 2.5 times more fluorescent signal than cells incubated with LS. LGS are internalized 2.5 times more than LS: this fact shows that the cellular bioavailability of LGS is greater than that of LS. b) Comparative cell pharmacology of free lipogelosomes® -PA / PA:

A. AZT and 3TC, effects on macrophages in culture: In these experiments, LGS encapsulating AZT or 3TC were incubated with human macrophagic cells. The cytotoxicities of the free or encapsulated products in the LGS were analyzed. The results show that the encapsulated PAs are 150 times more effective than free AZT or 3TC, in terms of cytotoxicity with respect to macrophages in culture.

These experiments show that at equal doses the encapsulated PA is 150 times more active vis-à-vis the macrophages than the free PA, probably due to its better cellular internalization.

B. Doxorubicin and PEG 4000, effects on hepatocytes and differentiated intestinal epithelial cells, in culture:

We compared the cellular internalization of two molecules: doxorubicin and PEG 4000, according to whether their form is free or encapsulated in the form of LGS.

Under the same experimental conditions in time and concentration, the fact that the molecule is encapsulated causes an increase in its cellular incorporation or in its pharmacological activity with respect to cells of hepatic or intestinal origin, by a factor varying from 1.5 to 3.

These experiments show that the bioavailability of molecules encapsulated in the form of LGS is increased compared to their free form on intestinal epithelial cells or on hepatic parenchymal cells. c) Explanation of the modified bioavailability of molecules when they are in the form of LGS:

Liposomes (LS) are usually internalized at the cellular level by a process of membrane fusion, called passive diffusion, that is to say not involving secondary messengers responsible for the expression of a membrane receptor. However, LGS differs from LS by the presence of a particular thermoreversible gel in the internal phase of LGS, irradiating to the surface of the particle as well as by the presence of a gel film on its external surface. of proteo-saccharic nature (mixture of gelatin, carrageenans K and i).

The differences in cellular internalizations and therefore in bioavailability between the LS and the LGS according to the invention essentially reside in the presence and the composition of this gel. Differentiated or undifferentiated intestinal cells

(strains: HT29, HT29gal, T84) were cultured on a semi-porous filter (diffusion chamber). LGS were incubated with these cells in the presence or absence of cpt-cAMP. In the presence of cpt-cAMP, the internalization of LGS is increased by a factor of 2.

This experiment shows that the internalization of LGS is a cAMP-dependent phenomenon. Furthermore, the internalization curve for LGS as a function of dose shows that the phenomenon of internalization of LGS is saturable. These two essential facts show that the internalization of LGS is mediated by a receptor. This internalization process therefore differs from endocytosis processes by fusion of conventional liposomes, which are not dependent on a receptor. Thus the optimized bioavailability of drugs encapsulated in LGS is explained by the use of a specific LGS receptor. d) Bioavailability of the lipogelosome® forms in vivo in rats: see example 3 e) Bioavailability of the lipogelosome® forms on the diffusion chamber model:

Intestinal epithelial cells were cultured at confluence on a semi-porous filter, making it possible to obtain a two-compartment system, separating a "plasma" medium from an "intestinal lumen" medium. LGS were placed on the “intestinal lumen” side, then after an incubation period, the medium

"Plasma" was analyzed.

In this compartment, we find by laser granulometry structures having the same characteristics as the LGS deposited at the start of the experiment in the “intestinal lumen” compartment. The addition of a proportion of CDCA (chenodeoxycholate) in the formulation of LGS, increases the number of particles found in the "plasma" compartment.

These experiments show that a proportion of LGS crosses the intestinal epithelium, presumably by a process of paracellular passage or transcytosis. This passage is increased when the LGS formulation is modified by the addition of CDCA. Thus, it is possible to optimize the intestinal transepithelial passage

(after oral administration, for example) of molecules encapsulated in

LGS; the LGS form makes it possible to increase the intestinal bioavailability of the encapsulated molecules, this property is exacerbated when the chenodeoxycholate is included in the LGS formulation.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims

CLAIMS 1°) Liposomal vectors of active ingredients, characterized in that they consist of: - a powder composition which is essentially made up of unilamellar liposomes comprising an external lipid phase consisting of class 4 lipids (phospholipids), possibly associated with substances class 2 (long-chain triglycerides, cholesterol esters), class 3 substances (cholesterol, non-ionized long-chain fatty acids) and/or class 5 substances (bile salts, fusidic acid derivatives) and an internal aqueous core forming a thermoreversible aqueous gel irradiating to the external lipid phase, which internal aqueous core consists essentially of a mixture M of at least two non-polymerizable gelling agents Gl and G2, different and whose point of gel-sol phase transition is greater than or equal to 37°C, Gl being a gelling agent selected from gelatins and carrageenans and G2 being selected from carrageenans having properties different from carrageenans selected for Gl, and celluloses, which liposomes have a diameter between 20 nm and 1 mm, preferably between 20 nm and 500 nm and which is in the form of particulate units having an average diameter between 10 mm and 1000 mm, formed of a or several of said liposomes, surrounded by a matrix selected from the group consisting of a dehydrated thermoreversible aqueous gel identical to the aqueous gel of said internal core, dextrins or a mixture thereof, such that it comprises, in average, 1016 to 1018 liposomes/g of powder and - at least one active ingredient included, depending on the case, either in the gelled internal core or in the external lipid phase. 2°) Liposomal vectors of active ingredients according to claim 1, characterized in that they further comprise, in their internal aqueous core, at least one stabilizing agent of osidic nature, and/or at least one agent for regulating the osmolarity of the medium and/or at least one surfactant, such as a bile salt and/or a non-ionic surfactant. 3°) Liposomal vectors of active ingredients according to claim 1 or claim 2, characterized in that they comprise in % (m/m): 25 to 75% of class 4 lipids, 5 to 45% of gelling agents , 0 to 70% of stabilizing agent of osidic nature, 0 to 15% of agent for regulating the osmolarity of the medium, 0 to 20% of surfactants and 0 to 15% of dextrins. 4°) Liposomal vectors of active ingredients according to any one of claims 1 to 3, characterized in that said aqueous internal core comprises 70 to 95% of gelling agent Gl and 5 to 30% of gelling agent G2. 5°) Liposomal vectors of active ingredients according to any one of claims 2 to 4, characterized in that the stabilizing agent of osidic nature is sucrose, trehalose or any other protective agent. 6°) Process for preparing powder vectors according to any one of claims 1 to 5, in which the external matrix of the particulate units comprises a fraction of dehydrated thermoreversible aqueous gel, characterized in that it comprises the following steps:

(1) preparation of a dispersion of liposomes with a gelled inner core (lipogelosomes ^® ) in an aqueous phase by (a) preparation of a solution of at least one suitable gelling agent, in particular a mixture M of gelling agents Gl and G2 , by dissolving said gelling agents, with slow stirring, at a temperature higher than the gel-sol phase transition temperature of said gelling agents in an aqueous solution at a pH compatible with the active principle to be encapsulated, (b) incorporation of the active principle into the solution obtained in (a), (c) incorporation of the lipids into the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum and formation of an emulsion and (d ) obtaining said dispersion of liposomes with a gelled internal core (lipogelosomes ^® ) in an aqueous phase containing said gelling agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum and

(2) obtaining the powder product by suitable drying of the dispersion obtained.

7°) Method according to claim 6, characterized in that the drying is carried out by atomization, coacervation, thin layer or granulation. 8°) Process for preparing powder vectors according to any one of claims 1 to 5, in which the external matrix of the units particles comprises a thermoreversible aqueous gel fraction and/or a dextrin, characterized in that it comprises the following steps:

(1) preparation of a dispersion of liposomes with a gelled inner core (lipogelosomes ^® ) in an aqueous phase by (a) preparation of a solution of at least one suitable gelling agent, in particular a mixture M of gelling agents Gl and G2 , by dissolving said gelling agents, with slow stirring, at a temperature higher than the gel-sol phase transition temperature of said gelling agents in an aqueous solution at a pH compatible with the active principle to be encapsulated, (b) incorporation of the active principle into the solution obtained in (a), (c) incorporation of the lipids into the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum and formation of an emulsion and (d ) obtaining said dispersion of liposomes with a gelled internal core (lipogelosomes ^® ) in an aqueous external phase containing said gelling agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum, (2) elimination of at minus a part of the aqueous liquid phase containing said gelling agents, in which the liposomes are dispersed,

(3) addition of at least one suitable dextrin and

(4) obtaining the powder product by spray drying the product obtained in (3). 9°) Method according to claim 8, characterized in that step (2) of eliminating at least part of the aqueous liquid phase containing said gelling agents is carried out by dilution and/or by filtration.

10°) Method according to any one of claims 6 to 9, characterized in that the aqueous solution of step (a) further comprises an agent for regulating the osmolarity of the medium (NaCl at 0.9 %, for example) and/or a stabilizing agent of an osidic nature and/or a surfactant, preferably class 5 substances (bile salts).

11°) Preparation process according to any one of claims 6 to 10, characterized in that step (b) of incorporation of the active ingredient is carried out in the external lipid phase, before incorporation of the latter into the mixture obtained in (a). 12°) Method according to any one of claims 6 to 11, characterized in that step (c) is carried out, preferably, at a shear speed of less than 200 s ^"1 .

13°) Pharmaceutical composition, characterized in that it comprises a liposomal vector of active principle according to any one of claims 1 to 5 and at least one pharmaceutically acceptable vehicle.

14°) Composition according to claim 13, characterized in that it further comprises a cAMP activator.

15°) Composition according to claim 13 or claim 14, characterized in that it is in solid form (capsule, tablet, powder to dissolve in water).

16°) Dispersion of liposomes with gelled internal cores in an aqueous solution containing the mixture of gelling agents as defined in claim 1, in which said liposomes have the following morphology: . vesicular structure with a diameter of between 20 nm and 500 nm, preferably between 20 and 80 nm, and

. polydispersity of liposomes with gelled internal phase of between 10 and 55%, preferably between 10 and 30%.