WO2007014754A1

WO2007014754A1 - Process for the preparation of liposomal formulations

Info

Publication number: WO2007014754A1
Application number: PCT/EP2006/007606
Authority: WO
Inventors: Peter Fankhauser; Gérard Riess; Pascal Breton; Jacques Bartholeyns
Original assignee: I.D.M. Immuno-Designed Molecules
Priority date: 2005-08-02
Filing date: 2006-08-01
Publication date: 2007-02-08
Also published as: EP1909758A1

Abstract

The invention relates to a homogenous powder comprising or consisting of: 0.01 - 20% (w/w) of one or several amphiphilic substances with solubility Parameters δd comprised between 15.0 and 23.0 J1/2/cm3/2 and (δp2 + δh2)1/2 comprised between 6.0 and 13.0 J1/2/cm3/2, 80 - 99.99% (w/w) of a combination of lipids consisting of: i. 65 - 95% (w/w) of palmitoyl-oleoyl-phosphatidylcholine (POPC), ii. 5 - 35% (w/w) of dioleoyl-phosphatidylserine (DOPS), iii. 0 - 20% (w/w) of other phospholipids, 0 - 10% (w/w) of cholesterol, and further characterized by: having a specific volume of 4 - 40 ml/g of blended lipids, showing in the differential scanning calorimetry (DSC) tracing a Single endothermic transition with a monomodal peak with a maximum at 30 - 50°C and a transition enthalpy of 10 to 30 J/g.

Description

PROCESS FOR THE PREPARATION OF LIPOSOMAL FORMULATIONS

The invention relates to a new process for preparing liposomal formulations, in particular, liposomal suspensions.

The invention also relates, to a homogeneous powder containing synthetic phospholipids, possibly cholesterol, and one or several amphiphilic substance(s) of biological interest, which can be dispersed in an aqueous medium to give a liposomal suspension, and to pharmaceutical compositions containing said homogeneous powder. The invention also relates to a dispersion containing synthetic phospholipids, possibly cholesterol, and one or several amphiphilic substance(s) of biological interest which can be used, in particular, as an intermediate product for preparing said homogeneous powder.

The present invention also relates to methods for preparing said homogeneous powder and said dispersion and to in vivo methods for activating the immune system. Different types of liposomes (small or large, unilamellar or multilamellar) have been described in the literature, in numerous publications, for various uses. Liposomes consist of one or more micrometric and concentric spheres of neutral and/or electrically- charged lipids forming molecular bilayers, which surround an internal cavity containing or entrapping an aqueous phase. These liposomes may contain a compound, possibly one or several biologically active substances, which are present in the internal aqueous phase and/or embedded in the lipid bilayers, depending on their molecular structures, their resulting physico-chemical characteristics, their respective concentrations and other liposome manufacturing parameters such as temperature.

In therapeutic treatments, liposomes are used to conveniently formulate active substances in order to improve their pharmacokinetic profile, their biodistribution and their bioavailability, to target specific tissues and/or cell population and, finally, to reduce their toxicity while increasing their efficiency. Besides their ability to improve the therapeutic index of encapsulated biologically active compounds, liposomes present important advantages such as the reduction of the efficient dose of formulated biologically active substances as compared to the use of the same free compounds.

Several documents relate to methods for the preparation of liposomes: US patent 6,066,331 (Barenholz and coll., Opperbas Holding B. V.) describes a method for the encapsulation of biological structures, biopolymers or oligomers (biologically active agents) into lipid membrane vesicles. This method of preparation is based on two separated steps of solubilization, wherein lipids, i.e. amphiphilic materials such as dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG) are solubilized in a polar solvent miscible with water (tertiary-butanol hereafter also named t- butanol)(solution A) and the biologically active agent is dispersed in a physiologically compatible aqueous medium possibly containing a cryoprotectant (solution B). Solution A and solution B are then mixed. Therefore, according to this method the amphiphilic substance of biological interest is initially not present in the t-butanol phase but only in the aqueous medium.

US patent 6,156,337 (Barenholz and coll., Opperbas Holding B. V.) describes another method for preparing loaded liposomes wherein lipids (amphiphilic substances) are mixed in a water-immiscible organic solvent (such as fluorinated hydrocarbons, chlorinated hydrocarbons), and said solvent is then removed in the presence of a solid support. The dried lipids are placed into an aqueous solution of the biopolymeric substances (biologically active agent) in a physiologically compatible solution which contains a cryoprotectant. Then, an organic solvent is added, preferentially t-butanol, before performing the lyophilization. US patent 4,971,802 (Tarcsay and coll., Ciba-Geigy Corporation) relates to lyophilization of a solution containing two synthetic phospholipids and a substance or a mixture of substances having biological activity in an organic solvent, especially t-butanol.

The freezing temperature of t-butanol is 25.5°C (CRC Handbook of Chemistry and

Physics 51th Edition). At this temperature or below, the use of t-butanol in an industrial application raises some problems such as clogging of feed lines and filling equipment. The use of t-butanol in an industrial process implies to work in a warm chamber, at a controlled temperature permanently higher than about 25-26°C at which, also, the prevention of working staff from t-butanol vapours is difficult.

One of the aims of the invention is to provide a new process for preparing liposomal formulations, said process being suitable to be implemented on an industrial scale. Thanks to the homogeneous powder obtained as an intermediate, all the liposomes of the resulting liposomal suspensions are of uniform composition. The suspensions do not contain sub- populations with different compositions.

Another aim of the invention is to provide a new homogeneous powder consisting of one or several amphiphilic substance(s) of biological interest, preferentially a lipophilic immunostimulant and a combination of phospholipids, with or without cholesterol, useful for in vivo activation of the immune system, with said homogeneous powder being suitable to be used for in vivo administration to possibly form, in the human organism, liposomes which might be equivalent/similar to liposomes prepared in vitro.

Another aim of the invention is to provide a dispersion as an intermediate product for the preparation of said homogeneous powder.

Another aim of the present invention is to provide a method for preparing said dispersion and another method for preparing said homogeneous powder comprising a step of lyophilizing said dispersion.

Another aim of the invention is to provide in vivo methods, in particular, for activating the immune system, especially, in order to cure and/or prevent cancer or infectious diseases.

All those aims have been achieved by the present invention which in its broadest embodiment, provides a new process for preparing liposomal formulations, this process comprising a dispersion step (followed by a lyophilization step) of phospholipids, eventually cholesterol, and one or several amphiphilic substances of biological interest in an appropriate blend of solvents, allowing an industrial production of a dispersion from which can be obtained a homogeneous powder and, subsequently, a liposomal suspension. The Inventors surprisingly observed that a particular blend of solvents consisting of water and t-butanol used in this invention has, in particular, favourable dispersion properties for amphiphilic lipids.

The present invention relates to a homogeneous powder comprising or consisting of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δ_d is comprised between 15.0 and 23.0 J^1/2/cm^3/2 and the parameter (δ_p ² + δ_h ²)^I/2 is comprised between 6.0 and 13.0 J'^/cm^3/2,

• 80 to 99.99% (w/w) of a combination of lipids consisting of: i. 65 to 95% (w/w) of palmitoyl-oleoyl-phosphatidylcholine (POPC), ii. 5 to 35% (w/w) of dioleoyl-phosphatidylserine (DOPS), iii. 0 to 20% (w/w) of other phospholipids,

• 0 to 10% (w/w) of cholesterol, and further characterized by:

• having a specific volume of 4 to 40 ml/g of blended lipids, and

• showing in differential scanning calorimetry (DSC) tracing a single endothermic transition with a monomodal peak showing a maximum at 30- 5O⁰C and a transition enthalpy of 10-30 J/g. The Inventors found that a blend of lipids comprising l-palmitoyl-2-oleoyl- phosphatidylcholine (POPC) and dioleoyl-phosphatidylserine (DOPS), does precipitate from f-butanol dispersion upon the addition of 1% (w/w) and up to 10% (w/w) of water. The Inventors surprisingly observed that this phospholipid composition is still fully dispersible when more than 10% (w/w) water is added to the solvent. This unexpected phenomenon occurs in a particular embodiment of the invention with a POPC:DOPS favorable blend of 7:3 (weight ratio), and more particularly when an amphiphilic substance of biological interest is present in the blend of lipids. This is in contradiction to the expectations on dispersibility behavior for those skilled in the art.

The lyophilized powders obtained by freeze-drying such dispersions are homogeneous as demonstrated by DSC (Differential Scanning Calorimetry) and form, in contact with an aqueous medium, liposomes that are homogeneous with respect to their morphology, as can be shown by size measurement, or to their composition, as can be shown by FFE (Free Flowing Electrophoresis).

This observation has possibly to do with the formation of nanostructures in t- butanol blends. The formation of such nanostructures can be shown by light diffraction studies as explained below. It appears again surprisingly that the homogeneity of nanostructures is narrow in t-butanol dihydrate and deteriorates outside of the range according to this invention, which comprises /-butanol-dihydrate and blends of f-butanol- dihydrate with an excess of up to 40% (w/w) of additional /-butanol. This phenomenon can be shown by the Polydispersity Index determined by light scattering measurements of particle size, (as shown in Example 12). Until now, it has never been undertaken to replace /-butanol by a solvent freezing at a temperature much lower than room temperature in order to conveniently prepare liposomes at industrial scale, in particular, in the case that the active substance is not soluble in an aqueous medium. Whereas such lipids are dispersed in 100% of /-butanol, they precipitate already at low water concentrations of 1 - 10%. It is reminded that water and /-butanol form /-butanol dihydrate (i.e. a complex comprising one molecule of /-butanol associated with two molecules of water) and depending on the respective concentrations of water and /-butanol, there remains more or less free water or /-butanol in excess. It leads to a blend of /-butanol dihydrate and water, or to a blend of /-butanol dihydrate and /-butanol, hereafter designed as blends of solvents.

The composition of the /-butanol dihydrate in stoichiometric conditions contains 67.3% (w/w) of /-butanol and 32.7% of water (w/w). As an approximate value, a composition comprising 67.3 ± 1% (w/w) of /-butanol and 32.7 ± 1% (w/w) of water will be considered as corresponding to 100% /-butanol dihydrate. As an approximate value, the composition of the /-butanol dihydrate in stoichiometric conditions contains preferably 68.1% (w/w) of /-butanol and 31.9% of water (w/w).

Furthermore, in an unexpected manner, the Inventors have shown that the amphiphilic substances of biological interest are dispersed in a blend of /-butanol dihydrate and /-butanol more efficiently than in a solvent based on 100% of /-butanol.

The major industrial interest of the present invention, is that the use of a blend of solvents prepared from /-butanol and water provides a cheaper, more convenient and more robust industrial production of liposomal formulations than prior art methods which used a solvent based on 100% of /-butanol, or a mixture of two organic solvents. For a number of reasons, including difficulty in handling 100% /-butanol (solid at room temperature) as well as concerns related to industrial hygiene (high vapor pressure of /-butanol might lead to high worker's exposure), current requirements at commercial manufacturing facilities appear to totally preclude the use of 100% /-butanol as the solvent to be lyophilized.

By adding water to /-butanol, the freezing temperature of /-butanol is dramatically decreased. Besides, the major difficulty consisting of not disturbing, by addition of water, the dispersion of the combination of phospholipids, possibly cholesterol and amphiphilic substances of biological interest, which are simultaneously dispersed in the same solvent, is unexpectedly overcome.

By "homogeneous powder" is meant an essentially dry substance or an essentially dry composition of substances, in form of a porous cake of low density or of loose powder formed from this cake by mechanical impact, in which all parts present the same characteristics. Said homogeneous powder is devoid of contaminants (such as organic solvents, metal or glass particles as e.g. from sonication methods, surfactants as e.g. from dialysis methods) allowing their use in human therapy. This homogeneous powder may contain residual water (0.1 to less than 5%), tightly bound to the lipids, resulting from the process for preparing said powder.

The expression "amphiphilic substance of biological interest" designates all the compounds such as biological or chemical molecules, which contain both hydrophobic (or non polar) and hydrophilic (or polar) groups.

The amphiphilic substance is preferentially a substance with a biological activity. Its means that said substance could interact, in vitro or in vivo, with a component of animal or human cells and induce a response after said interaction. Substances having biological activity may be pharmaceutical substances such as immuno-activators, antibiotics, antidepressants, anti-inflammatory drug, drugs for respiratory or cardiovascular system, for cancer or all the other diseases. In a particular embodiment, the amphiphilic substance of biological interest is an anionic or a non-ionic substance. More particularly, the amphiphilic substance may be chosen among the group consisting of anionic substances, such as JBT3002, MTP-PE, sodium tetradecylsulfate, amphotericine B, or of non-ionic substances, such as sitosterol, dehydroepiandrosterone, pregnenolone and hydropolyethoxydodecane. In another particular embodiment, the amphiphilic substance is an anionic substance. More particularly, the amphiphilic substance may be chosen among the group consisting of: JBT3002, MTP-PE, sodium tetradecylsulfate and amphotericine B.

The expression "other phospholipids" designates phospholipids which are selected from the group of: natural or synthetic glycerol derivatives in which two of the hydroxyl groups of glycerol are linked through an ester bond to fatty acids e.g. myristic, stearic, palmitic, oleic, linolic, linoleic, arachidonic, elaidic acid or through an ether bond to fatty alcohols and the third hydroxyl group is linked to phosphoric acid or through a phosphoric acid ester linkage to a polar head group such as an amino acid, e.g. serine, a sugar e.g. inositol, an amino alcohol e.g. choline or ethanol amine or others as found in natural compounds of this class. In a particular embodiment, other phospholipids are chosen among the group consisting of: 1 ,2-Di-Oleoyl Phosphatidyl Glycerol (DOPG), l-Palmitoyl-2-Oleoyl Phosphatidyl Glycerol (POPG), 1 ,2-Di-Palmitoyl Phosphatidyl Ethanolamine (DPPE).

The expression "specific volume of blended lipids" designates the bulk volume used by each gram of the combination of lipids.

The expression "solubility parameters" designates for a given substance the intrinsic physical value, accessible by different theories, that allows to predict its solubility in a particular solvent and its miscibility or compatibility with another component.

The most widely used theory allowing to estimate by calculation the solubility parameters of a substance is that of Hoftyzer - Van Krevelen (D. W. Van Krevelen: "Properties of Polymers" Chap. 7, 3^rd Edition, Elsevier Publ. 1990). According to these authors, the total solubility parameter δ_t of a substance is defined such as: δ_t ² = δ_d ² + δ_p ² + δ_h ² where: δd is the dispersive or apolar contribution δ_p is the polar contribution δ_h is the hydrogen bonding contribution, the units being in J^1/2 / cm^3/2 or (MPa)^1/2.

For amphiphilic substances, it is more suitable to take into account, on the one side the apolar contribution δ_d and on the other side, the total polar contribution which is generally defined by the geometrical mean value:

The solubility parameters of amphiphilic substances of biological interest

(dipalmitoylphosphatidyl choline, sodium tetradecylsulfate, pregnenolone, dehydroepiandrosterone (DHEA), sitosterol, muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) (US 4 971 802), bis-(taurine)-L-glutaminyl-N-PalmitoyI-S-[2-(R)-3- dilauroyloxypropyl]-L-cystine, C₅₅Hi₀₂N₄O₁₄S₃Na₂, M_w = 1,185 (JBT3002) (US 5 342 977), calculated with this method are summarized in the Table I. Table I: Solubility parameters of amphiphilic substances of biological interest.

Differential Scanning Calorimetry (DSC) ("Differential Scanning Calorimetry" J.H. Flynπ in Encyclopedia of Polymer Science and Engineering 2nd edition, Chapter Thermal Analysis, p. 690-723. Wiley-Interscience 1989) is a method to investigate the presence of ordered/crystalline ranges of material within a sample. The Inventors demonstrate that DSC is a suitable technique to determine the homogeneity of a powder according to the invention, by its thermal characteristics, and that these thermal characteristics (i.e. number of transition peaks, onset and peak maximum, enthalpy of the transition) are reproducible data. From DSC thermograms, it can be seen whether a solid combination of lipids is homogeneous with respect to its crystalline structure. A monomodal transition peak with a well-defined maximum shows that only one type of homogeneous material is present, whereas a bimodal peak with 2 distinguishable maxima or 2 separate peaks underlines a phase separation into more than one type of structure. Transition enthalpy is the energy required to change a substance from a state to another (solid-liquid transition) such as from a crystalline solid into an amorphous solid or liquid.

In a particular embodiment of the present invention, the homogeneous powder comprises or is constituted by: • 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δ_d is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm³*

• 80 to 99.99% (w/w) of a combination of lipids consisting of: i. about 70% (w/w) of POPC ii. about 30% (w/w) of DOPS and further characterized by :

• having a specific volume of 10 to 40 ml/g of blended lipids,

• and showing in the DSC tracing a single endothermic transition with a peak maximum at 30 - 50°C and a transition enthalpy of 10 to 30 J/g. The homogeneous powder according to the invention shows in the DSC tracing, a single endothermic transition with a peak maximum at 30-45°C.

The powder described is stable and can under a protective gas, as e.g. nitrogen or argon, be stored frozen or at temperatures up to 8⁰C for many years or at room temperature (< 25°C) for several months. The homogeneous powder results in particular from the lyophilization of a dispersion which will be referred to hereafter.

The invention also relates to a dispersion of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δd comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 80 - 99.99% (w/w) of a combination of lipids consisting of 65 — 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed at a concentration of 2.5 - 10 % (w/w) in a blend of 60 to 100% (w/w) of /-butanol-dihydrate and 0 to 40% (w/w) of /-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of f-butanol.

The use of t-butanol dihydrate enables a manufacturer to an efficient production of lyophilizates without the need for special temperature-controlled room and piping conditions that are commonly not available at pharmaceutical manufacturer facilities. The process using 100% /-butanol has to be conducted as follows: ^■=> phospholipid dispersion in f-butanol is prepared at 50⁰C in a sealed vessel; ^■=> the dispersion is then filtered at 50°C through a sterile filter (0.2 μm) linked, through a thermostatic piping, to the vessel into the filter housing contained in a secondary thermostatic container; it is then kept at 35°C into a second thermostatic storage vessel;

<=> dispensing into vials is done with a standard vial filling equipment in a room heated to 30⁰C in order to avoid solidification of f-butanol in the piping and the piston pumps; ^ filling machine-housing room is vented with a venting equipment in order to keep t-butanol vapors below an acceptable working place concentration. The process with J-butanol dihydrate is conducted as it follows: ^■=> phospholipid solution in r-butanol is prepared at 50⁰C in a sealed vessel. The solution is allowed to cool to 20 - 25°C; <=> the solution is filtered through a standard sterile filter (0.2 μm) into a standard storage container; "=> vial filling is performed using a standard filling machine in a standard room of a pharmaceutical production plant.

By "dispersion" is meant a mixture of two or more substances forming a colloidal dispersion (from about 1 to about 1,000 nm) into a liquid continuous phase. When colloid size is less than 1 to 2 nm, the dispersion is referred to as a molecular dispersion also called solution.

By "solution" is meant a liquid mixture of two or more substances forming a continuous phase at the molecular level. The invention also relates to a dispersion of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δ_d is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2, • 80 to 99.99% (w/w) of a combination of lipids consisting of 65 to 95% (w/w) of POPC, 5 to 35% (w/w) of DOPS, 0 to 20% (w/w) of other phospholipids,

• 0 to 10% (w/w) of cholesterol, dispersed at a concentration of 2.5 - 10% (w/w) in a blend of 60 to 100% (w/w) of t- butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of f-butanol-dihydrate and 0 to 25% (w/w) of t-butanol, and characterized in the temperature range of 20 to 30°C by an average particle size below 4.0 nm, and preferably below 3.0 ± 0.5 nm with a polydispersity index of less than 1.1, and more preferably of less than 1.05. This corresponds typically to a nanoscale colloidal system.

A particle size distribution is characterized by the mean values (average values): number mean diameter (NMD), volume mean diameter (VMD) and the polydispersity in size is usually characterized by the ratio VMD/NMD (polydispersity index, PI). A value of

1.00 or close to 1.00 signifies that all particles have the same size, the higher the deviation from 1.00 is, the higher the size polydispersity is.

In a particular embodiment, the invention relates to a dispersion of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δj is comprised between 15.0 and 23.0 J^I/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^l/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 80 to 99.99% (w/w) of a combination of lipids consisting of 65 to 95% (w/w) of POPC, 5 to 35% (w/w) of DOPS, 0 to 20% (w/w) of other phospholipids,

• 0 to 10% (w/w) of cholesterol, dispersed at a concentration of about 7% (w/w) in a blend of 60 to 100% (w/w) of t- butanol-dihydrate and 0 to 40% (w/w) of f-butanol, preferably in a blend of 75 to 100% (w/w) of /-butanol-dihydrate and 0 to 25% (w/w) of /-butanol.

In another particular embodiment, the invention relates to a dispersion of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s), for which the solubility parameter δa is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2, • 80 to 99.99% (w/w) of a combination of lipids consisting of 65 to 95% (w/w) of POPC, 5 to 35% (w/w) of DOPS, 0 to 20% (w/w) of other phospholipids,

• 0 to 10% (w/w) of cholesterol, dispersed at a concentration of about 7% (w/w) in a blend of about 91.7 ± 1% t-butanol- dihydrate and of about 8.3 ± 1% of t-butanol.

For instance, the size of colloidal suspension can be measured by static or dynamic light scattering methods or ultracentrifugation or small angle X-ray scattering (SAXS) or small angle neutron scattering (SANS). Such dispersions can be handled at room temperature and are stable for several days.

The expression "t-butanol" designates tert-butanol, as well as tertzo-butanol or tertiary butanol, an organic solvent.

The expression "t-butanol-dihydrate" designates tert-butanol-dihydrate, as well as tert/o-butanol-dihydrate or tertiary butanol-dihydrate, a compound resulting in the close and stable association between one (1) molecule of /-butanol and two (2) molecules of water.

It is well known from literature (Ott et al. ^Thermodynamics 11, p.739-746 (1979); Woznyj et al., Z.Naturforsch. 40a, p. 693 - 698 (1985); Kasraian et ai., Pharmaceutical Research Vol. 12, No. 4, (1995), p. 484 - 490)) that t-butanol and water form molecular complexes leading to a phase diagram of the t-butanol/water system with two distinct regions separated by a relative maximum of the melting point at a composition corresponding to a dihydrate of t-butanol. Areas of lowered melting point (with melting point minima called eutectics) exist for compositions between pure water and the t-butanol dihydrate and between t-butanol and the t-butanol dihydrate. The t-butanol-dihydrate stoichiometrically contains 67.3% of t-butanol and 32.7% of water (w/w). It may be admitted that t-butanol-dihydrate contains 67.3 ± 1% of t-butanol and 32.7 ± 1% of water (w/w), and in this case, t-butanol-dihydrate stoichiometrically preferably contains 68.1% of t-butanol and 31.9% of water (w/w).

In the present invention, the solvent can be added either in the form of t-butanol- dihydrate (prepared before the use by mixing suitable quantities of t-butanol and water), or in the form of t-butanol and water mixed with all other components of the homogeneous powder according to the invention. In a particular embodiment, the solvent is first prepared by mixing the desired quantities of t-butanol and water, therefore forming t-butanol- dihydrate, then dispersing the lipids in the obtained solvent. The other components mostly do not impede the formation of t-butanol-dihydrate.

The blend of solvents based on 67.3% of t-butanol and 32.7% of water (w/w) (stoichiometric conditions) allows to obtain about 100% of t-butanol-dihydrate (see Example 1).

If stoichiometric conditions are not observed, then water or t-butanol is in excess. For instance, a blend of solvents constituted by 60% of t-butanol and 40% of water (w/w), provides t-butanol dihydrate and water in excess. 60 g of t-butanol represent 0.81 mole, and 40 g of water represent 2.22 moles. But only 1.62 mole of water (0.81*2) could be complexed with the 0.81 mole of t-butanol. So 0.60 mole of water is in excess, which represents 10.8 g (see Example 4). The composition of the solvent is then of about 89.2% of t-butanol-dihydrate and about 10.8% of water.

Another example: a blend of solvents constituted by 70% of t-butanol and 30% of water (w/w) provides t-butanol dihydrate and t-butanol in excess. 70 g of t-butanol represent 0.94 mole, and 30 g of water represent 1.67 moles. Only 0.83 mole of t-butanol (1.67/2) couid be complexed with the 1.67 mole of water. So, O.i i mole of t-butanoi is in excess, which represents 8.15 g (see Example 4). The composition of the solvent is then of about 91.8% of t-butanol-dihydrate and about 8.2% of t-butanol. According to a preferred embodiment of the present invention, the solvent is constituted by a blend of 75 to 100% (w/w) of t-butanol dihydrate and 0 to 25% (w/w) of t- butanol. In other words, 75 to 100% (w/w) of t-butanol dihydrate can be obtained by mixing 67.3 to 75% (w/w) of t-butanol and water so as to complete to 100% (a range between 32.7 and 25% (w/w)). According to a more preferred embodiment of the present invention, the solvent is constituted by a blend of 88.2% of t-butanol dihydrate and 11.8% of t-butanol (w/w). In other words, 88.2% of t-butanol dihydrate can be obtained by mixing about 71.2% of t- butanol and about 28.8% water (w/w).

The dispersion of lipids in t-butanol-dihydrate can be preferentially achieved at about 50°C (see Example 2). Nevertheless, the dispersion of lipids can be also achieved, for instance, at room temperature. The dispersion of the lipids in t-butanol-dihydrate can be more preferentially achieved at a temperature comprised between about 2O⁰C to 40°C, and preferably below 25 ± 1°C. (see Example 2).

The invention also relates to a homogeneous powder described above such as the one prepared by a process comprising a step of lyophilizing a dispersion of: • 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2, • 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of

POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids, • 0 - 10% (w/w) of cholesterol , dispersed in a blend of 60 to 100% (w/w) of ^-butanol-dihydrate and 0 to 40% (w/w) of t- butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol.

In another embodiment, the present invention relates to a homogeneous powder described above, such as the one prepared by a process comprising: a) a step of preparing a dispersion comprising or consisting of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2, • 80 - 99.99% of a combination of lipids consisting of 65 - 95% (w/w) of

POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol, and b) a step of lyophilizing said dispersion.

In an advantageous embodiment of the invention, one amphiphilic substance of biological interest or at least one of the amphiphilic substances of biological interest contained in a homogeneous powder according to the invention is a selected amphiphilic immunostimulant. In an advantageous embodiment of the invention, the amphiphilic immunostimulant is associated with amphiphilic peptides or lipopeptide antigens (description hereafter). The association of one amphiphilic immunostimulant and one or more amphiphilic peptides or lipopeptide antigens within the homogeneous powder is designed to induce also specific immune responses to the amphiphilic peptides or lipopeptide antigens.

The expression "amphiphilic immunostimulant" designates all substances able to trigger innate immune responses via receptor such as TOLL and NOD receptors (Inohara N. and Nunez G., Nature Rev. Immunol., 2003, 3(5): 371-382) expressed in monocytes, macrophages, dendritic cells, NK cells or polynuclear cells, in vitro or in vivo, and able to get anchored, through its lipidic part, into the lipid bilayers of a liposome. Examples of amphiphilic immunostimulants are muramyl tripeptide phosphatidyl ethanolamine (MTP- PE), bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2-(R)-3-dilauroyloxypropyl]-L-cystine (JBT 3002), sitosterol, Lipid A or others LPS derivatives or amphiphilic CpG motif rich nucleotides. The present invention is not limited to the amphiphilic immunostimulants described above.

In a particular embodiment of the present invention, the amphiphilic immunostimuiant is muramyl tripeptide phosphatidyl ethanolamine (MTP-PE).

The muramyl tripeptide phosphatidyl ethanolamine has been described as an adjuvant for protective studies against tumor antigens or virus antigens (Herpes simplex virus or HIV- 1) (Burke RL., Rev Infect Dis., 1991, 13:S906-l 1; Obert M. and coll., Vaccine. 1998, 16(2- 3):161-9; Graham BS. And coll., Ann Intern Med., 1996, 125(4):270-9). MTP-PE has a stimulating effect on cell proliferation (Wachsmuth ED, Huber J., Virchows Arch B Cell Pathol Incl MoI Pathol., 1989;58(l):45-57) and able to activate the cytotoxic capabilities of monocytes (Galligioni E and coll., Int J Cancer. 1993, 55(3):380-5). In another particular embodiment of the present invention, the amphiphilic immunostimulant is JBT 3002.

Bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2-(R)-3-dilauroyloxypropyl]-L-cystine (JBT3002) is a synthetic bacterial lipopeptide (able to activate macrophages and induce production of inflammatory cytokines (TNF-α, IL-I, IL-6) (Kumar R. and coll., Cancer Biother Radiopharm., 1997, 12(5):333-40). In another particular embodiment of the present invention, the amphiphilic immunostimulant is sitosterol.

By sitosterol, is meant sitosterol, as well as όeta-sitosterol, όeta-sitosterol glucoside. The imrnunostimulating capacity of 6eta-sitosterol (a phytosterol) has been demonstrated in vitro and in vivo. The έeta-sitosterol is able to enhance T cell proliferation in presence of phytohaemagglutinin, to stimulate NK cells activity and induce an increased secretion of IL-2 and gamma- interferon by lymphocytes (Bouic P.J.D and coll., Int. J. Immunopharmac. 1996, 18(12): 693-700).

The amphiphilic immunostimulants as described above can be associated with amphiphilic peptides or lipopeptide antigens. Said amphiphilic peptides or lipopeptide antigens are preferably formed by peptidic chains of 8 to 16 amino-acids (considered as immunogenic peptides), linked via the NH₂ terminal group to a aliphatic and lipidic chain of 5 to 30 carbons, more preferentially of 8 to 18 carbons. Typical immunogenic peptides used are selected from wild or modified peptidic antigens with high affinity for the MHC class I and MHC class II molecules. Said peptides can be selected from the group consisting of CTL inducing peptides, tumor cell antigen peptides, or hepatitis antigen peptides. More preferentially said peptides are selected from the group consisting of carcinoma solid tumor cell antigens (SEQ ID N° 1 to 32 (WO 0142270), and 130 to 134 US 6.602.510, WO 01.45728 and US 07.976.301), melanoma antigens (SEQ ID N° 77 to 107; US 5.662.907 and US 5.750.395), hepatitis B or C antigens (SEQ ID N⁰ 135 to 151) or other tumor cell antigens such as breast cancer antigens 5T4 (SEQ ID N° 124 to 129; WO 03068816), Her2/neu antigens (SEQ ID N° 33 to 76; US 2004 157780) or p53 antigens (SEQ ID N° 108 to 123; WO 00141787).

A combination of 2 to 10 different amphiphilic peptides or lipopeptide antigens mixed in a powder according to the invention, in a dispersion thereof, or in a resulting liposomal suspension are able to induce innate or specific immune responses to those lipopeptidic antigens. More preferentially, these amphiphilic peptides or lipopeptide antigens are covalently linked to the T helper PADRE peptide also known as pan-DR epitope peptide (SEQ ID N° 152). This epitope is known to improve immune responses in inducing a HTL (helper T cells) response supporting the induction of antibody responses (Alexander J. and coll., Vaccine, 2004, 22:2362-7; Franke ED. and coll., Vaccine, 1999, 17: 1201-5). This peptide is described in US 5.736.142 and US 6.413.935 patents.

In a particular embodiment, an amphiphilic protein such as KSA (Epithelial Cell Adhesion Molecule) rather than the polypeptides described above, is incorporated in the phospholipid suspension together with amphiphilic immunostimulant. The resulting lyophilisate powder is used to induce an immune response to this protein.

In another advantageous embodiment of the invention, one amphiphilic substance or at least one of the amphiphilic substance(s) is selected from the group of hormones preferentially from the group of steroid hormones such as dehydroepiandrosterone (DHEA) or pregnenolone.

Contrarily to Cortisol, the other adrenal glucocorticoid known to suppress the immune response, several studies show the stimulatory effect of the dehydroepiandrosterone (DHEA) on immune system. As described by Khorram and collaborators, DHEA activates T lymphocytes and provides an increased number of monocytes, B cells and NK cells in age-advanced men (Khorram O. and coll., J. Gerontol. A Biol. Sci. Med. Sci., 1997, 52(1): Ml-7). Another effect of DHEA is to induce monocyte-derived DC differentiation in presence of GM-CSF/IL-4 to produce immature DC (Canning M.O. and coll., Eur. J. of Endocrinology, 2000, 143: 687-695).

According to a particular embodiment of the present invention, a homogeneous powder as described above has a phase transition at body temperature (36-40⁰C).

The invention also relates to a method for preparing the dispersion described above comprising: a) a step of preparing of a mixture of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δ_d is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 0.5 to 10% (w/w) of cholesterol b) a step of dispersing said mixture into a solvent consisting of a blend of 60 to 100% of t-butanol-dihydrate and 0 to 40% of t-butanol, preferably in a blend of 75% to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol.

Said method containing a step of dispersing said mixture into a solvent consisting of a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t- butanol, allows to decrease the freezing temperature of t-butanol from 27°C to below 5°C.

This strongly facilitates the use of this solvent as compared to pure t-butanol in an industrial process. Said industrial process can be performed without the need of heated rooms or chambers.

The invention also relates to a method for preparing the dispersion described above comprising: a) a step of preparing a mixture of:

• 0.01 to 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δd is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 0 to 10% (w/w) of cholesterol, b) a step of preparing a solvent consisting of a blend of 60 to 100% (w/w) of t- butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol, c) a step of dispersing said mixture into said solvent.

The invention also relates to a method for preparing a homogeneous powder according to the invention, comprising the step of lyophilizing a dispersion comprising or consisting of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δd is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2, * 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of /-butanol-dihydrate and 0 to 40% (w/w) of t- butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of /-butanol.

The invention also relates to another method for preparing said homogeneous powder. This method comprises: a) a step of preparing a dispersion comprising or consisting of: • 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest, for which the solubility parameter δ_d is comprised between 15.0 and 23.0 J^1/2/cm^3/2, the parameter (δ_p ² + δ_h ²)^1/2 is comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of /-butanol-dihydrate and 0 to 40% (w/w) of t- butanol, preferably in a blend of 75 to 100% (w/w) of f-butanol-dihydrate and 0 to 25% (w/w) of J-butano 1. b) a step of lyophilizing said dispersion.

In a particular embodiment of the present invention, the method for preparing a homogeneous powder according to the invention consists of a lyophilization of the dispersion described above, which is subjected to the following steps: - fill the dispersion into vials,

- place vials onto the shelf of a lyophilizer,

- flush the lyophilizer with argon,

- cool down for 2 hours to -3O⁰C and keep 2 hours at -30⁰C, at atmospheric pressure, - evacuate to 0.01 mbar and sublimate for 2 hours, - warm up for 10 hours up to -10⁰C and keep for 5 hours at -10⁰C, at 0.01 mbar,

- warm up for 3 hours up to 20⁰C and maintain at a temperature of 2O⁰C and a pressure of 0.01 mbar for 2 hours flush with argon and close the vials under argon.

The invention also relates to a multi-lamellar liposomal suspension obtained by contacting a homogeneous powder according to the invention with an aqueous medium. The aqueous medium is suitable for therapeutic purposes, namely sterile, with physiological pH and possibly with preservatives or anti-oxidants. If necessary, the liposomal suspension is buffered to pH 7.0 - 7.5. The average particle size of the liposomes is between 2 and 8 μm, preferentially between 4 and 6 μm. All liposomes are identical with respect to their composition. The liposomal suspensions are stable at 4°C for several days.

The amphiphilic substance of biological interest encapsulated in a liposome is preferably associated with the phospholipids constituent of liposomes and therefore located within the lipid bilayers of the liposomes, but can also be partly present in the interior space of the liposome.

The invention also relates to the use of a homogeneous powder or a multi-lamellar liposomal suspension according to the invention for the in vivo activation of the immune system.

This activation of the immune system is achieved by uptake of the liposomal suspension by immuno-competent cells which are then activated following the binding of the immunostimulant amphiphilic substance to specific receptors. This activation can also be obtained via an initial ex vivo step of activation in culture conditions of specific immuno-competent cells such as monocytes, macrophages or dendritic cells.

To the use of said powder for the in vivo activation of the immune system for the treatment of animal or human patients suffering from cancer or infectious diseases, all components of the homogeneous powder can be used in a sterile form.

According to a particular embodiment of the invention, a homogeneous powder or a multi-lamellar liposomal suspension are used for the treatment of animal or human patients suffering from cancer or infectious diseases. According to another particular embodiment of the invention, a homogeneous powder or a multi-lamellar liposomal suspension are used for the prevention of cancer recurrence or recurrence of infectious diseases.

The invention also relates to an in vivo method for activating the immune system comprising the step of contacting a homogeneous powder according to the invention, with immune cells through oral or local administration.

Oral administration designates an administration by ingestion of tablets, pills or caps of the powder, the dispersion according to the invention. Local administration designates a pulmonary or tonsils administration by dry spray or a transdermal administration by a needle free device.

According to a particular embodiment of the above mentioned method for activating the immune system, the homogeneous powder is micronized to an average size of less than 2 micrometers, for inhalation or transdermal application.

For inhalation application, the homogeneous lyophilized powder according to the invention is loaded into a dry spray device to deliver said homogeneous lyophilized powder in the form of an aerosol. Said dry spray device allows to deposit said homogeneous iyophiiized powder for example into the throat, on tonsiis, or advantageously directly into the pulmonary alveolae where resident macrophages can be directly activated by liposomal suspension formed in situ. The invention also relates to an in vivo method for achieving sustained hormonal level following the local administration of hormones contained in the homogeneous powder according to the invention.

The invention also relates to a pharmaceutical composition containing as active substance the homogeneous powder, according to the invention, in association with a pharmaceutically acceptable vehicle.

In a preferred embodiment, the pharmaceutical composition according to the invention contains the homogeneous powder present in a range of 50mg to 2g in a single application or unit.

The invention also relates to the use of a homogeneous powder or a dispersion according to the invention for the preparation of an immunostimulating drug. The colloidal characteristics of POPC/DOPS blends dispersed in t-butanol-water mixtures can be determined as follows:

To characterize the phospholipid blends, such as POPC/DOPS blends and those containing an additional lipid component dispersed in t-butanol-water mixtures, the inventors performed DLS measurements. These measurements were carried out in order to determine the average particle size of the dispersed species. From this characteristic it could be defined if the dispersion of phospholipids leads to a solution, also called molecular dispersion, or to a colloidal suspension if the particle size is greater than 1-2 nm.

For colloidal systems it is of interest to determine the polydispersity index of the dispersion. In fact, a particle size distribution is characterized by the mean values (average values): number mean diameter (NMD), volume mean diameter (VMD) and the polydispersity in size is usually characterized by the ratio VMD/NMD (polydispersity index, PI). A value of 1.00 or close to 1.00 signifies that all particles have the same size, the higher the deviation from 1.00 is, the higher the size polydispersity is. The NANOTRAC technique is based on the analysis of the Brownian movement of the dispersed particles in a liquid by acquisition of the energy spectrum corresponding to the Doppier shift.

The apparatus MTUPA 250 - NANOTRAC 250, equipped with a laser at 780 nm, operates by laser diffusion for particles in the size range from 0.8 to 6500 nm. It is ISO 13321 standard certified for granulomere determinations.

The NANOTRAC 250 has the following advantages:

- to operate in situ with very small volume samples (0.1 to 3 ml)

- to work without dilution even at high concentrations of dispersed material.

For the particle size determination, it is necessary to know the viscosity of the solvent at a given temperature.

The viscosities of the t-butanol-water mixtures were determined experimentally in the 20-30⁰C temperature range.

A literature search (CHEMINFO data bank) showed that the viscosity values for pure t-butanol are as follows: 4.31 centipoises (cp) at 25°C and 3.35 cp at 30°C. The viscosities of the t-butanol-water mixtures were determined experimentally in the 20-30°C temperature range, e.g. for t-butanol-dihydrate, it is 5.87 cp, 4.82 cp and 3.87 cp when determined at 20°C, 25°C and 30°C, respectively.

Description of the figures:

Figure 1 represents the chemical structure of bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2- (R)-3-dilauroyloxypropyl]-L-cystine (JBT 3002) (C₅₅Hi₀₂N₄Oi₄S₃Na₂).

Figure 2 represents a typical DSC profile (obtained for the powder containing 70% POPC and 30% DOPS without further amphiphilic components). The Y-axis represents the heat flow (mW) and the X-axis represents the temperature (⁰C). The scanning rate is of

20.0°C/min and the weight of the sample tested is 5.970 mg. The peak of temperature is from 23.57 to 47.43°C with a maximum at 38.77°C, an onset at 35.39°C and a transition enthalpy of 23.87 J/g. The peak around 100⁰C is not a direct characteristic of the material, it can be attributed to the vaporization of a very small amount of water.

Figure 3 represents the DSC profile of the powder from Example 2. The Y-axis represents the heat flow (mW) and the X-axis represents the temperature (⁰C). The scanning rate is of 20.0°C/min and the weight of the sample tested is 5.880 mg. The peak of temperature is from 25.63 to 50.27⁰C with a maximum at 39.74⁰C, an onset at 36.25⁰C and a transition enthalpy of 24.27 J/g. The peak around 100⁰C is not a direct characteristic of the material, it can be attributed to the vaporization of a very small amount of water.

Figure 4 represents the sequences of the amphiphilic peptides or lipophilic antigens previously referred to in the description.

Examples

Example 1: Preparation of t-butanol-dihydrate for laboratory use

7ert-butanol (TBA) 99.5% (melting point 25-26°C, water content < 0.03%) was purchased from ACROS. Ultrapure water (purified by ion-exchange and inverse osmometry - Millipore) was used for dilution.

1.682 kg of t-butanol are melted by warming at 30°C in a 21 screw cap bottle, 0.818 kg of water are added under stirring and the t-butanol-dihydrate gets formed. It is kept in the tightly closed bottle for further use. t-butanol-dihydrate can be used at room temperature or even under refrigeration in dosing and handling devices without risk of solidification.

Example 2: Preparation of a lyophilized powder containing a amphiphilic immunostimulant on industrial level

A 100 1 barrel of t-butanol was warmed to 30°C for 24 hours by placing the barrel in a heated room. 57.790 kg t-butanol were filled into a stainless steel vessel with a heating jacket. 28.10 kg of purified water for injection were added under stirring to form t-butanol- dihydrate. The temperature of the blend was raised to 50°C and 25 g of MTP-PE (muramyl tripeptide phosphatidyl ethanolamine), 4.375 kg of l-palmitoyl-2-oleoyl phosphatidyl choline (POPC) and 1.875 kg of 1,2-dioleoyl phosphatidyl serine (DOPS) (corresponding to a combination of 70% POPC and 30% DOPS), were added under stirring. Stirring was continued for 1 hour in order to fully disperse the components. 7.830 kg of t-butanol was added under stirring to obtain the optimal industrial blend.

The preparation was cooled down to room temperature (20 - 25°C), filtered through a standard sterile filter (0.2 μm) into a standard storage container. This container was transported to a sterile manufacturing area. There it was attached, through a standard sterile filter, to a standard vial filling machine. 6,000 vials of 70 ml volume were filled with 16.0 g (density: 0.783) of the dispersion. The filled vials were placed on stainless steel trays and those were placed in a production shelf lyophilizer Usifroid. The lyophilization procedure was implemented under the following conditions: freeze down for 2 hours to -30°C at atmospheric pressure, keep for 2h at this temperature and at atmospheric pressure, then, reduce the pressure up to 0.01 mbar, warm up within 1Oh to -10°C, keep for 5h at -1O⁰C, warm up within 3h to 20⁰C and dry for 2h.

Example 3: Analysis by Differential Scanning Calorimetry (DSC)

5 to 15 mg (precisely weighed to 0.01 mg) of the lyophilized powder of Example 2 (in the form of a homogeneously looking opaque, white cake) were filled into a Differential Scanning Calorimetry (DSC) pan Ref-BO14-3017 - 50 μl cover-lid B014-3003 (Perkin-

Elmer) at room temperature and under atmospheric pressure conditions (760 mm Hg). The

DSC pans were closed with the cover lid punctured with 2 small holes in order to avoid a pressure building-up during the heating cycles.

The pan was placed in a DSC Perkin-Elmer Model 7. The instrument was calibrated daily using a reference compound, cyclohexane (peak onset -87.1⁰C; peak max -84.7°C; ΔH =

80.5 J/g) and indium (peak onset 157.2⁰C ; peak max 158.7°C; ΔH = 27.9 J/g).

The thermograms were recorded by first cooling the sample from roorn temperature to -90° at a constant rate of 20°C/min, followed by heating to +12O⁰C at the same constant rate. A typical recording is given in Fig.3.

The DSC performed on the lyophilized powder of Example 2 shows a single peak with an onset at 35.0 ± 1.0⁰C, a maximum at 38.0 ± 1.5°C and a transition enthalpy of 22.0 ± 2.5 J/g.

Example 4: Effect of the solvent blend on lyophilized powder characteristics

1,050 mg (1.05 g) of l-palmitoyl-2-oleoyl phosphatidyl choline (POPC) and 450 mg of 1,2- dioleoyl phosphatidyl serine (DOPS), corresponding to a combination of 70% POPC and 30% DOPS, were dispersed in 22.5 g of solvent of a composition as given in Table II, heated to 50⁰C and stirred until fully dispersed. 4.0 g of the dispersion were filtered from a syringe directly through a 0.22 μm filter cartridge into each of 5 vials of 15 ml volume.

The lyophilization was performed in a Heraeus GT 4 lyophilizer under the conditions given in the Example 2. Table II: Lyophilization from different blends of solvents.

The percentage of each component is given with a possible standard deviation of 1%.

All samples after lyophilization formed opaque, white, homogeneously looking cakes of approximately 4 ml.

The Differential Scanning Calorimetry (DSC) was performed as given in Example 3 and tracings showed the following characteristics:

Table HI: DSC of lyophilized powders (homogeneous or not) resulting from different blends of solvents.

Samples D, E and F represent the most robust mixtures of solvent for homogeneous powders with monomodal DSC peaks. B, C and G mixtures do not reproducibly produce homogeneous powders of the invention. The optimal composition for homogeneity is in the range claimed by this application.

Example 5: Effect of the lipid ratio (POPC / POPS) on lyophilized powder characteristics l-palmitoyl-2-oleoyl phosphatidyl choline (POPC) and 1,2-dioleoyl phosphatidyl serine (DOPS) as specified in Table IV were dispersed in 22.5 g of a blend of 91.7% (w/w) of t- butanol-dihydrate and 8.3% (w/w) of f-butanol, heated to 50°C and stirred until dispersed.

4.0 g of the dispersion were filtered from a syringe directly through a 0.22 μm filter cartridge into each of 5 vials of 15 ml volume.

The lyophilization was performed in a Heraeus GT 4 lyophilizer under the conditions given in Example 2.

Table IV: Lyophilized powders (homogeneous or not) with different lipid compositions.

The Differential Scanning Calorimetry (DSC) was performed as given in Example 3 and tracings showed the following characteristics: Table V: DSC of lyophilized powders (homogeneous or not) with different lipid compositions.

The range including samples K through N represents the most robust mixtures of lipids for homogeneous powders with monomodal DSC peaks. This means that a content of DOPS between 5 and 35% gives the most advantageous powder.

Example 6: Effect of an amphiphilic substance of biological interest on lyophilized powder characteristics

1,050 mg of l-palmitoyl-2-oleoyl phosphatidyl choline (POPC) and 450 mg of 1,2-dioleoyl phosphatidyl serine (DOPS) and various amounts of muramyl tripeptide phosphatidyl ethanolamine (MTP-PE) were dispersed in 22.5 g of a blend of 91.7% (w/w) of f-butanol- dihydrate and 8.3% (w/w) of t-butanol, heated to 50⁰C and stirred until dispersed. 4.0 g of the dispersion were filtered from a syringe directly through a 0.22 μm filter cartridge into each of 5 vials of 15 ml volume.

The lyophilization was performed in a Heraeus GT 4 lyophilizer under the conditions given in Example 2. Table VI; Lyophilized powders (homogeneous or not) with different contents of amphiphilic substances of biological interest.

For POPC the following values apply: δ_d : 16.8

(δ_p ^z ₊ δV 2T_Λl/2. : 7.2 r rl/27/cm -3/2

The Differential Scanning Calorimetry (DSC) was performed as given in Example 3 and tracing showed the following characteristics:

Table VII; DSC of lyophilized powders (homogeneous or not) with different contents of amphiphilic substances of biological interest.

From the results, it can be seen that the lipid lyophilizates remain homogeneous with various amphophilic substances of biological interest up to therapeutic concentrations.

Example 7: Effect of cholesterol on lyophilized powder characteristics

Cholesterol is frequently used as a co-lipid in liposomes, especially to modulate the lipid bilayer rigidity.

1,050 mg of l-palmitoyl-2-oleoyl phosphatidyl choline (POPC) and 450 mg of 1,2-dioleoyl phosphatidyl serine (DOPS) and various amounts of cholesterol were dispersed in 22.5 g of a blend of 91.7% (w/w) of f-butanol-dihydrate and 8.3% (w/w) of f-butanol, heated to 50⁰C and stirred until dispersed. 4.0 g of the dispersion were filtered from a syringe directly through a 0.22 μm filter cartridge into each of 5 vials of 15 ml volume.

Table VIII; Effect of cholesterol on lyophilized powders.

Both samples after lyophilization formed opaque, white, homogeneously looking cakes of approximately 4 ml.

Table IX: DSC of lyophilized powders containing different amount of cholesterol.

The addition of cholesterol leads to a lowering of the temperature at peak onset and maximum.

Example 8: Effect of other phospholipids on lyophilized powder characteristics

5 mg of MTP-PE and the components according to Table X were weighed into a vial, dispersed at 50°C in 2.75 g of t-butanol-dihydrate, filtered through a 0.22 μm filter and filled into sterile, particle-free vials of 15 ml volume. 4 Vials were filled with 4.0 g of the dispersion, the rest of the dispersion was used to measure light scattering measurements. The 4 vials were frozen into a freezer at - 24°C for 24 h. Lyophilization was done in a laboratory lyophilizer chamber. The vials maintained at -24°C were introduced into the chamber cooled at -5°C and immediately placed under vacuum. After 5 h of lyophilization, the chamber was allowed to warm up to room temperature within 8 h. After additional 8 h under vacuum at room temperature, the chamber was warmed up at 30°C and the materials allowed to dry for 4 h.

Table X: Composition of lyophilised powders containing additional phospholipids.

The Differential Scanning Calorimetry (DSC) was performed as given in Example 3 and tracing showed the following characteristics: Table XI: DSC of lyophilized powders with different contents of other phospholipids.

Example 9: Liposomal suspensions

50 ml of sterile saline solution (0.9% NaCl in water for injection) were aspirated through a spike-filter into a syringe, and introduced through the spike-filter into a vial from Example 2. While keeping the spike-filter with the syringe in place, the sterile saline solution was allowed to stand in contact with the powder for 1 min in order to allow hydration of the combination of lipids, then the vial with filter and syringe still in place was vigorously shaken for 1 min and the formed liposome suspension aspirated through the spike-filter into the syringe. From there, it can be directly infused using an electrically driven syringe pump or it can be injected into an infusion bag and infused from there by gravity feeding.

Example 10: Liposome size determination by Fraunhofer diffraction 1 ml of the liposomal suspension of Example 9 was diluted with 40 ml of particle free NaCl solution 0.9% (w/w). A diffraction photometer, Sympatec Helos, (Sympatec GmbH, Germany) with flow through cell of 50 mm focal length was filled with particle free 0.9% NaCl solution (filtered twice through a pressure filter device fitted with a 0.2 μm filter). The solution was circulated with the peristaltic pump of the instrument. Using a Pasteur pipette, the diluted suspension was transferred dropwise into the tank of the circulating NaCl solution, such that the instrument reading reached an optical concentration of 5%. Stirrer speed: 40 - 60 % pump speed: 60 - 80% duration of the measurement: 20 s

Mean diameter: 5.8 μm

Percentage between 1.5 and 10.0 μm: > 77.6% Percentage smaller than 1.5 μm: < 2.5 % Percentage larger than 10.0 μm: < 20.2 %

The liposomes were of multi-lamellar nature as visible by light microscopy in polarized light.

Example 11: Reduction of liposomal size

The suspension formed in Example 9 was collected with a syringe through a filter holder fitted with a filter with straight line pores of nominal diameter of 3 μm, while allowing air to enter the vial through a hydrophobic filter.

As measured by Fraunhofer diffraction and as described in Example 10, the liposomes formed had the following size characteristics:

Mean diameter: 4.7 μm

Percentage between 1.5 and 10 μm: 90.7% Percentage smaller than 1.5 μm: < 3.0% Percentage bigger than 10 μm: < 6.6%

Example 12: Colloidal characteristics of phospholipid blends dispersed in t- butanol-water mixtures

Particle size determination of phospholipid blends dispersed in f-butanol-dihvdrate

Several phospholipid blends as prepared in the Examples 2, 4 and 8 were dispersed in t- butanol-dihydrate (67.5% /-butanol / 32.5 water weight ratio) at 50°C during 15 min and then slowly cooled down to 30°C, then to 25°C and possibly to 20°C, which corresponds to the temperature range where the viscosities of the solvent systems have been determined experimentally. TABLE XII: Composition of the samples used for characterization of their solutions in /-butanol-dihydrate.

The results (average of 5 measurements) are given in Table XIII.

TABLE XIII: Particle size (volume average VMD in nm) and polydispersity index (PI) of phospholipid blends in /-butanol-dihydrate.

Phospholipid concentration: 7% weight ratio with respect to solvent

10 For the VMD values given with an accuracy of ± 0.1 nm, it can be noticed that the particle size of all these samples is in the range of 2.4 to 3.4 nm, which is characteristic for nanoscale colloidal systems. The interesting feature is further that the polydispersity index is low, PI between 1.02 and 1.08 which is an indication that the nanoscale colloidal systems are almost monodispersed in size.

This particle size range is indirectly in agreement with those obtained by dynamic light scattering (DLS) with a COULTER N4 Plus instrument supplied by COULTRONICS. With this device it could be shown that the particle size of the above mentioned phospholipid dispersions is below 3 to 4 nm, which corresponds to the theoretical lower size detection limit of this technique.

Particle size determination on phospholipid blends dispersed in f-butanol-dihydrate or in pure f-butanol

To compare the effect of dispersion in r-butanol-dihydrate and in pure /-butanol, the same phospholipid blends were dispersed in /-butanol-dihydrate or in 100% t-butanol. The results are given in Table XIV, from which it appears clearly that in both cases we have nanoscale colloidal systems, however, with the major difference that the dispersion of the phospholipids in pure t-butanol leads to higher particle sizes and polydispersity indices with respect to /-butanol-dihydrate.

TABLE XIV: Particle size (volume average VMD in nm) and polydispersity index (PI) of phospholipid blends in pure f-butanol or in f-butanol-dihydrate.

This means that, in pure /-butanol, colloidal suspensions are significantly more polydispersed in size than in J-butanol-dihydrate.

An additional feature, which is advantageous for the freeze drying process, is that the onset of precipitation for the lipids is distinctly shifted to lower temperatures for many lipid compositions when operating in t-butanol-dihydrate instead of pure t-butanol. This comparison is shown in Table XV below.

TABLE XV: Precipitation temperature of phospholipids in /-butanol-dihydrate and in /-butanol.

(**) solid formation (freezing of t-butanol) below 12°C (in undercooled solvent) (* * *) evolution with the number of thermal cycles

The comparison t-butanol-dihydrate versus pure f-butanol has shown that the particle size and the polydispersity index (PI) of the phospholipid dispersions are influenced by the solvent characteristics. In order to confirm this tendency, other systems were studied, which are either within or beyond the claim composition limits. The results are given in Table XVI below.

TABLE XVI: Particle size (volume average VMD in nm) and polydispersity index (PI) of phospholipid blends in f-butanol-water mixtures of different compositions.

Phospholipid concentration: 7 % (weight ratio).

One can notice from these tests, carried out in the temperature range of 20 to 30°C with different lipid compositions - that there is a tendency of particle size increase with increasing t-butanol contents, and

- that the polydispersity index reaches minimum values in the claimed solvent composition range of the t-butanol-dihydrate.

Nanoscale colloidal systems with the lowest polydispersity indices, e.g. particles of almost same size, can thus be obtained in the claimed solvent composition range. Example 13: Effect of liposomal suspensions on human macrophages

In vitro, immuno-potentiators, as exemplified by MTP-PE, induce TNF-α secretion by human macrophages, as indicator of macrophage activation. Serial dilutions of liposomal suspension containing MTP-PE (from Example 9) were prepared by adding to said suspension Ultra-glutamine (Biowhittaker, Cat n° BE 12- 702F/Ul)-containing RPMI 1640 cell culture medium such that MTP-PE concentrations were in the range 0.0025 - 50 μg/ml. Ultra-glutamine-containing RPMI 1640 cell culture medium was used as a negative control and a 1 μg/ml LPS solution was used as a positive control.

One hundred microliters of the test solution were added to the wells of a 48-flat-bottom well plate. One hundred microliters of human macrophages differentiated from monocytes (7 days incubation with GM-CSF, according to a process described in WO 94/26875 and WO96/0029) at 2 x 10⁶ cells/ml in test medium (RPMI 1640 + 10% FCS) were introduced to each well of (2 x 10⁵cells/well) with a multichannel pipette. Macrophages were tested for cell viability, purity and antigen cell surface expression.

After 18h (± 30 min) of incubation at 37⁰C, in 5% CO₂ atmosphere, cell cultures were centrifuged at 1,200 rpm for 5 min at room temperature to pellet macrophages. One hundred and fifty microliters of cell culture supernatants were collected with a multichannel pipette and transferred to microtubes and frozen at -80⁰C. After a freezing/thawing step, cells were tested for cell viability, cell recovery, antigen cell surface expression, sterility and ability to secrete TNF-α following culture with macrophage activators. TNF-α measurements were performed on macrophage supernatants according to an established protocol "Cytokine (TNF-α) Measurement by ELISA", using appropriate dilutions series of the supernatants as required by this protocol. Quantikine human TNF-α (R&D Systems, Cat n° STAOOC) was used as the reference standard. 50% effect concentrations were calculated according to generally established methods.

^■=> TNF-α secretion by macrophages Macrophages were cultured overnight in two separate plates in presence of a dose range of MTP-PE liposomal formulation and one dose of LPS as a positive control. Supernatants were harvested and tested for TNF-α concentration. MTP-PE liposomal formulation lots induced a dose dependent TNF-α secretion from macrophages.

^■=> Statistical analysis of TNF-α secretion dose responses Median Effective Concentration (EC50) for TNF-α secretion was determined by fitting a sigmoidal dose-response model to experimental data using non-linear regression. This model is the classical 4-parameters dose-response model ("Hill model") combined with toxicity effects (2 parameters).

TNF-α dose responses observed following activation of thawed macrophages cell bank by dose ranges of MTP-PE liposomal formulation lots were fit to the 6-parameter model using non linear regression.

The EC50 best-fit values determined by the 6-parameters model were 0.776 μg/ml for the MTP-PE liposomal formulation lots. The variation index for EC50 determinations were below 17.1%.

Example 14: Inhalation powder Solid lyophilizate from Example 2 was passed through a jet mill to give an average particle size of 4 μm. The lyophilizate powder was loaded into a dry spray device for local or profound dispersion as an aerosol. It can equivalently be used in a powder inhalation device. The local spray of the lyophilizate allowed to reach the throat, tonsils while deep spray allowed to reach peripheral lung alveolae. This was first shown by a Mass Media Aerodynamic Diameter study, demonstrating the transport efficacy of inhalated particles on impactors, according to European Pharmacopea.

^■=> Physical distribution of inhaled lyophilized powder

Two lyophilized powders of phospholipids (Example 2) obtained according to the invention and containing 0.4% and 10% MTP-PE, respectively, were tested for their distribution after inhalation.

The powder inhalers tested were respectively Foradil (Novartis) and Relenza (GSK). The fine particles distribution was measured according to European pharmacopoea 2005 with a Glass Twin Impinger consisting of three segments: mouth and throat, STl segment mimicking oesophagus and large lung ducts, ST2 segment mimicking deep lung alveolae. The inhaled lyophilized powder accumulated mainly in the mouth and throat section, then in oesophagus and large lung section, and very little in deep alveolae.

A micronisation of the lyophilysed powder was necessary for inhalation since mean particle size was on average close to 20 μm before micronization and 4 μm after micronisation was performed using air jet mill HOSOKAWA A 550 Pharma on Ig product. The lyophilizate powder melted at body temperature in the organs reached and reconstituted liposomes in contact of water and fluids present locally.

It was shown in rodent models that these reconstituted liposomes were taken up by alveolar or tonsil macrophages (also tonsil dendritic cells).

The adjuvants (MTP-PE: 0.1 to 10 mg, amphiphilic peptides or lipopeptides: 0.1 to 5 mg for each peptide) contained in these in vivo reconstituted liposomes achieved macrophage activation as demonstrated by tests performed as described in Example 13 with macrophages recovered from lung iavage or tonsils scraping.

When in vivo experiments were performed with homogeneous powder containing a lipopeptide adjuvant and an amphiphilic peptide or a lipopeptide antigen specific to tetanus toxoid, it was also shown that the antigen presenting cells collected in vivo 2Oh" after the spray, were able to induce ex vivo T cells proliferation and TNFα secretion by T lymphocytes specifically responding to tetanus toxoid.

This indicated that dry spray of the homogeneous powder containing immunostimulating adjuvants and lipophilic antigens can activate phagocytic cells (monocytes, macrophages and dendritic cells) to induce innate immune response (protection against the exogenous or abnormal antigens) and adaptive T cell response (specific to the lipophilic antigens). Example 15: Injectable powder / microsranulate

5 g of powder from Example 2 was formulated and micronized by spray-drying. For doing so, it was first solubilized or dispersed at 60°C in 4.4 1 ethanol. In parallel, 1.31 g lactose monohydrate was dissolved in 1.85 1 water. Both solutions were then mixed together. The powder is sprayed with a Niro Atomizer Portable spray drier (Columbia, MD). Compressed air with variable pressure (1-5 bars) ran a rotary atomizer located above the dryer with a drying air flow rate of 98 kg/h. Spray-dried particles were collected with a 6-inch cyclone. The inlet temperature was fixed at 110°C, the outlet temperature was about 46°C a V24 wheel rotating at 20,000 rpm was used, and the feed rate of the solution was 70 ml/min. The resulting micro-granules have an average particle size of 10-20 μm and are suitable for direct injection into skin using a needle-free powder injector, e.g. PowderJect ND device (PowderJect Vaccines, Madison WI), loaded with a 1 mg cassette-housed drug-containing powder. Two applications of those microgranules containing stable homogeneous powder with amphiphilic substance are tested.

The first application uses the normal DHEA incorporated into the liposome layer, its needled free injections as solid powder through human skin allows a slow release of the 100 μg DHEA content for optimal bio availability. The systemic concentration achieved and maintained during at least 24 h reach the levels claimed to prevent post menopausal problems, particularly osteoporosis in woman. This mode of administration of amphiphilic hormones appears adequate to achieve very prolonged and stable systemic biodistribution.

Example 16: Tablets

In a fluidized bed granulator type Glatt CPCG 1, 100 g of Avicel PH 102 and 20 g of lyophilizate from Example 2 (content of 20 vials) were fluidised at 40°C. 20 ml of a solution of 1 g of polyvinylpyrollidone (Plasdone K-25) were sprayed onto the powder for 30 min. The granules obtained were mixed in a Turbula mixer with 1O g of milled lactose and 1 g of magnesium stearate and compressed into tablets. In the current example, lyophilizate containing 1 mg of JBT 3002 was used to form tablets given orally to mice having been irradiated for 5 consecutive days and having developed mucositis. After receiving JBT 3002-loaded tablets, it was observed that severity of mucositis was markedly decreased with stimulation of intestinal epithelial cell growth.

Example 17: Effect of protein

1 vial of dry powder containing MTP-PE according to Example 2 is dispersed in 10 g of /- butanol. 4 aliquots of 0.7 g of this solution are filtered at 35°C through a sterile filter of 0.2 μm pore size into clean and sterile vials of 2 ml volume. 200 μl of a protein solution containing 0.5 mg/ml KSA in 0.9% sodium chloride are added to vials heated to 35°C and mixed by swirling the vials. The 4 vials are frozen into a freezer at -24°C for 24 h. Lyophilization was done in a laboratory lyophilizer chamber. The vials maintained at - 24⁰C were introduced into the chamber cooled at -5°C and immediately placed under vacuum. After 5 h of lyophilization, the chamber was allowed to warm up to room temperature within 8 h. After additional 8 h under vacuum at room temperature, the chamber was warmed up at 30°C and the materials allowed to dry for 4 h.

All samples after lyophilization form opaque, white, homogeneously looking cakes of approximately 1 ml. DSC trace shows the sample to be monomodal.

Claims

1. A homogeneous powder comprising or consisting of: • 0.01 - 20% (w/w) of one or several amphiphilic substances of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 80 - 99.99% (w/w) of a combination of lipids consisting of: i. 65 - 95% (w/w) of palmitoyl-oleoyl-phosphatidylcholine (POPC), ii 5 - 35% (w/w) of dioleoyl-phosphatidylserine (DOPS), iii 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, and further characterized by:

• having a specific volume of 4 - 40 ml/g of blended lipids, • showing in the differential scanning calorimetry (DSC) tracing a single endothermic transition with a monomodal peak with a maximum at 30 - 50°C and a transition enthalpy of 10 to 30 J/g.

2. The homogeneous powder of claim 1, comprising or consisting of: • 0.01 - 20% (w/w) of one or several amphiphilic substances of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^l/2/cm^3/2,

• 80 - 99.99% (w/w) of a combination of lipids consisting of: i. about 70% (w/w) of POPC, ii. about 30% (w/w) of DOPS, and further characterized by:

• having a specific volume of 10 - 40 ml/g of blended lipids,

• showing in the DSC tracing a single endothermic transition with a peak maximum at 30 - 50⁰C and a transition enthalpy of 10 - 30 J/g.

3. A dispersion of:

• 0.01 — 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 0 - 10% (w/w) of cholesterol, dispersed at a concentration of 2.5 - 10% (w/w) in a blend of 60 to 100% (w/w) of t- butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of /-butanol.

4. A dispersion according to claim 3 wherein the blend is of about 100% (w/w) of t-butanol-dihydrate.

5. A dispersion according to claim 3 or 4, said dispersion being characterized at 30°C by a polydispersity index of less than 1.1, and more preferably of less than 1.05.

6. A dispersion according to any one of claims 3 to 5 wherein the lipids are dispersed at a concentration of about 7% (w/w) in a blend of 60 to 100% (w/w) of t- butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of f-butanol-dihydrate and 0 to 25% (w/w) of /-butanol.

7. A homogeneous powder such as prepared by the process comprising a step of lyophilizing a dispersion of

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0

J^1/2/cm^3/2, • 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol.

8. The homogeneous powder of claim 7, such as prepared by the process comprising: a) a step of preparing a solution or a dispersion comprising or consisting of:

• 80 - 99,99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of r-butanol, preferably in a blend of 75 to 100% (w/w) of /-butanol-dihydrate and 0 to 25% (w/w) of t-butanol. b) b) a step of lyophilizing said dispersion.

9. The homogeneous powder of any one of claims 1 , 2, 7 or 8 wherein one or at least one of the amphiphilic substance(s) of biological interest is an amphiphilic immunostimulant.

10. The homogeneous powder of claim 9, wherein at least one amphiphilic substance(s) of biological interest is an amphiphilic immunostimulant associated with amphiphilic peptides or lipopeptide antigens.

11. The homogeneous powder of claim 9 or 10, wherein the amphiphilic immunostimulant is muramyl tripeptide phosphatidylethanolamine (MTP-PE).

12. The homogeneous powder of any one of claims 9 to 11, wherein the amphiphilic immunostimulant is bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2-(R)-3- dilauroyloxypropyl]-L-cystine (JBT 3002).

13. The homogeneous powder of any one of claims 9 to 11, wherein the amphiphilic immunostimulant is sitosterol.

14. The homogeneous powder of claim 10, wherein the amphiphilic peptides or lipopeptide antigens are selected from the group consisting of carcinoma solid tumor antigens, melanoma antigens, hepatitis B or C antigens.

15. The homogeneous powder of claims 1 or 2, wherein one or at least one of the amphiphilic substances of biological interest is selected from the group of hormones, preferentially from the group of steroid hormones, such as dehydroepiandrosterone or pregnenolone.

16. The homogeneous powder according to any one of claims 1, 2, 7 to 14 having a phase transition at body temperature (36-40°C).

17. A method for preparing the dispersion of any one of claims 3 to 6, comprising: a) preparing a mixture of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δa comprised between 15.0 and 23.0 J^1/2/cm³^ and (δ_p ² + δ_h ²)'^ comprised between 6.0 and 13.0 J^/cm^3/2, • 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95% (w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol , b) dispersing said mixture into a solvent consisting of a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol.

18. A method for preparing the dispersion of any one of claims 3 to 6, comprising: a) preparing a mixture of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2, • 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95%

(w/w) of POPC, 5 - 35% (w/w) of DOPS, 0 - 20% (w/w) of other phospholipids,

• 0 - 10% (w/w) of cholesterol, b) preparing a solvent consisting of a blend of 60 to 100% (w/w) of t-butanol- dihydrate and 0 to 40% (w/w) of t-butanol, preferably in a blend of 75 to

100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol. c) dispersing said mixture into said solvent.

19. A method according to claim 17 or 18 wherein the solvent consists of a blend of about 100% (w/w) t-butanol-dihydrate.

20. A method for preparing a homogeneous powder of any one of claims 1 , 2, or 7 to 16 comprising the step of lyophilizing a dispersion comprising or consisting of:

• 0.01 - 20% (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^1/2 comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 0 - 10% (w/w) of cholesterol, dispersed in a blend of 60 to 100% (w/w) of t-butanol-dihydrate and 0 to 40% (w/w) of r-butanol, preferably in a blend of 75 to 100% (w/w) of /-butanol-dihydrate and 0 to 25% (w/w) of f-butanol.

21. The method of claim 20, comprising: a) a step of preparing a dispersion comprising or consisting of:

• 0.01 - 20 % (w/w) of one or several amphiphilic substance(s) of biological interest with solubility parameters δ_d comprised between 15.0 and 23.0 J^1/2/cm^3/2 and (δ_p ² + δ_h ²)^lβ comprised between 6.0 and 13.0 J^1/2/cm^3/2,

• 80 - 99.99% (w/w) of a combination of lipids consisting of 65 - 95 % (w/w) of POPC, 5 - 35 % (w/w) of DOPS, 0 - 20 % (w/w) of other phospholipids,

• 0 - 10 % of cholesterol, dispersed in a blend of 60 to 100% (w/w) of f-butanol-dihydrate and 0 to 40% (w/w) of /-butanol, preferably in a blend of 75 to 100% (w/w) of t-butanol-dihydrate and 0 to 25% (w/w) of t-butanol, and b) a step of lyophilizing said dispersion.

22. A multi-lamellar liposomal suspension obtained by contacting the homogeneous powder according to any one of claims 1, 2, 7 to 16, with an aqueous medium.

23. The use of a homogeneous powder according to any one of claims 1, 2, 7 to 16 or of the multi-lamellar liposomal suspension of claim 22 for the in vivo activation of the immune system, possibly via an initial ex vivo step of activation of specific immuno -competent cells such as monocytes, macrophages or dendritic cells.

24. An in vivo method for activating the immune system comprising the step of contacting a homogeneous powder according to any one of claims 1, 2, 7 to 16, with immune cells through oral or local administration.

25. An in vivo method for activating the immune system according to claim 24, wherein the homogeneous powder is micronized to an average size of less than 20 μm, preferentially less than 10 μm and more preferentially comprised between 2.0 and 5.0 μm, for inhalation or transdermal application.

26. An in vivo method for achieving sustained hormonal level following the local administration of hormones contained in the homogeneous powder of any one of claims 1, 2, 7 to 16.

27. A pharmaceutical composition containing as active substance the homogeneous powder according to any one of claims 1, 2, 7 to 16, in association with a pharmaceutically acceptable vehicle.

28. Use of a homogeneous powder according to any one of claims 1, 2, 7 to 16, of or a dispersion according to any one of claims 3 to 6 or of a liposomal suspension according to claim 20 for the preparation of an immunostimulating drug.

29. Use of a homogeneous powder according to any one of claims 1 , 2, 9 to

16, of or a dispersion according to any one of claims 3 to 6 or of a liposomal suspension according to claim 20 for the preparation of a drug for the treatment of cancer or infectious diseases.

30. Use according to claim 29 for the preparation of a drug for the prevention of cancer recurrence or infectious diseases.