EP1909758A1 - Procédé pour la préparation de formulations liposomiques - Google Patents

Procédé pour la préparation de formulations liposomiques

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Publication number
EP1909758A1
EP1909758A1 EP06762930A EP06762930A EP1909758A1 EP 1909758 A1 EP1909758 A1 EP 1909758A1 EP 06762930 A EP06762930 A EP 06762930A EP 06762930 A EP06762930 A EP 06762930A EP 1909758 A1 EP1909758 A1 EP 1909758A1
Authority
EP
European Patent Office
Prior art keywords
butanol
dihydrate
blend
comprised
dispersion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06762930A
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German (de)
English (en)
Inventor
Peter Fankhauser
Gérard Riess
Pascal Breton
Jacques Bartholeyns
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IDM Immuno Designed Molecules
Original Assignee
IDM Immuno Designed Molecules
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Publication date
Application filed by IDM Immuno Designed Molecules filed Critical IDM Immuno Designed Molecules
Priority to EP06762930A priority Critical patent/EP1909758A1/fr
Publication of EP1909758A1 publication Critical patent/EP1909758A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

  • 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.
  • liposomes small or large, unilamellar or multilamellar
  • 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.
  • 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.
  • 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.
  • 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.
  • DMPC dimyristoyl phosphatidyl choline
  • DMPG dimyristoyl phosphatidyl glycerol
  • 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.
  • lipids amphiphilic substances
  • a water-immiscible organic solvent such as fluorinated hydrocarbons, chlorinated hydrocarbons
  • 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.
  • 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
  • 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.
  • the present invention relates to a homogeneous powder comprising or consisting of:
  • solubility parameter ⁇ d is comprised between 15.0 and 23.0 J 1/2 /cm 3/2 and the parameter ( ⁇ p 2 + ⁇ h 2 ) I/2 is comprised between 6.0 and 13.0 J' ⁇ /cm 3/2 ,
  • 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,
  • POPC palmitoyl-oleoyl-phosphatidylcholine
  • DOPS dioleoyl-phosphatidylserine
  • 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).
  • nanostructures in t- butanol blends have 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).
  • composition of the /-butanol dihydrate in stoichiometric conditions contains 67.3% (w/w) of /-butanol and 32.7% of water (w/w).
  • 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.
  • the composition of the /-butanol dihydrate in stoichiometric conditions contains preferably 68.1% (w/w) of /-butanol and 31.9% of water (w/w).
  • 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.
  • a solvent based on 100% of /-butanol or a mixture of two organic solvents.
  • current requirements at commercial manufacturing facilities appear to totally preclude the use of 100% /-butanol as the solvent to be lyophilized.
  • 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.
  • 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.
  • the amphiphilic substance of biological interest is an anionic or a non-ionic substance.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • DOPG 1,2-Di-Oleoyl Phosphatidyl Glycerol
  • POPG l-Palmitoyl-2-Oleoyl Phosphatidyl Glycerol
  • DPPE 1 ,2-Di-Palmitoyl Phosphatidyl Ethanolamine
  • 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.
  • amphiphilic substances it is more suitable to take into account, on the one side the apolar contribution ⁇ d and on the other side, the total polar contribution which is generally defined by the geometrical mean value:
  • DSC Differential Scanning Calorimetry
  • 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.
  • 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 ⁇ d is comprised between 15.0 and 23.0 J 1/2 /cm 3/2 , the parameter ( ⁇ p 2 + ⁇ h 2 ) 1/2 is comprised between 6.0 and 13.0 J 1/2 /cm 3 *
  • 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 8 0 C for many years or at room temperature ( ⁇ 25°C) for several months.
  • a protective gas as e.g. nitrogen or argon
  • 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:
  • 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.
  • 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.
  • colloid size is less than 1 to 2 nm, the dispersion is referred to as a molecular dispersion also called solution.
  • 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:
  • 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).
  • NMD number mean diameter
  • VMD volume mean diameter
  • PI polydispersity index
  • the invention relates to a dispersion of:
  • solubility parameter ⁇ j is comprised between 15.0 and 23.0 J I/2 /cm 3/2
  • the parameter ( ⁇ p 2 + ⁇ h 2 ) l/2 is comprised between 6.0 and 13.0 J 1/2 /cm 3/2
  • the invention relates to a dispersion of:
  • 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).
  • SAXS small angle X-ray scattering
  • SANS small angle neutron scattering
  • t-butanol designates tert-butanol, as well as tertzo-butanol or tertiary butanol, an organic solvent.
  • 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.
  • 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).
  • 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.
  • 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.
  • water or t-butanol is in excess.
  • 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
  • 40 g of water represent 2.22 moles.
  • 1.62 mole of water (0.81*2) could be complexed with the 0.81 mole of t-butanol.
  • 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.
  • 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
  • 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.
  • 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.
  • 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)).
  • the solvent is constituted by a blend of 88.2% of t-butanol dihydrate and 11.8% of t-butanol (w/w).
  • 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 2O 0 C 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 ⁇ d comprised between 15.0 and 23.0 J 1/2 /cm 3/2 and ( ⁇ p 2 + ⁇ 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • TOLL and NOD receptors Inohara N. and Nunez G., Nature Rev. Immunol., 2003, 3(5): 371-382
  • 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.
  • MTP- PE muramyl tripeptide phosphatidyl ethanolamine
  • JBT 3002 bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2-(R)-3-dilauroyloxypropyl]-L-cystine
  • 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.
  • 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).
  • 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- ⁇ , IL-I, IL-6) (Kumar R. and coll., Cancer Biother Radiopharm., 1997, 12(5):333-40).
  • the amphiphilic immunostimulant is sitosterol.
  • sitosterol is meant sitosterol, as well as ⁇ eta-sitosterol, ⁇ eta-sitosterol glucoside.
  • the imrnunostimulating capacity of 6eta-sitosterol (a phytosterol) has been demonstrated in vitro and in vivo.
  • the ⁇ eta-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).
  • 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 NH 2 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 N 0 135 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.
  • 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.
  • 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.
  • 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.
  • DHEA dehydroepiandrosterone
  • pregnenolone pregnenolone
  • DHEA dehydroepiandrosterone
  • a homogeneous powder as described above has a phase transition at body temperature (36-40 0 C).
  • the invention also relates to a method for preparing the dispersion described above comprising: a) a step of preparing of a mixture of:
  • solubility parameter ⁇ d is comprised between 15.0 and 23.0 J 1/2 /cm 3/2
  • parameter ( ⁇ p 2 + ⁇ h 2 ) 1/2 is comprised between 6.0 and 13.0 J 1/2 /cm 3/2
  • 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.
  • the invention also relates to a method for preparing the dispersion described above comprising: a) a step of preparing a mixture of:
  • solubility parameter ⁇ d is comprised between 15.0 and 23.0 J 1/2 /cm 3/2
  • the parameter ( ⁇ p 2 + ⁇ h 2 ) 1/2 is comprised between 6.0 and 13.0 J 1/2 /cm 3/2
  • 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:
  • 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 ⁇ d is comprised between 15.0 and 23.0 J 1/2 /cm 3/2 , the parameter ( ⁇ p 2 + ⁇ h 2 ) 1/2 is comprised between 6.0 and 13.0 J 1/2 /cm 3/2 ,
  • 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,
  • 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 ⁇ m, preferentially between 4 and 6 ⁇ m. All liposomes are identical with respect to their composition.
  • the liposomal suspensions are stable at 4°C for several days.
  • 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.
  • all components of the homogeneous powder can be used in a sterile form.
  • 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.
  • 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.
  • the homogeneous powder is micronized to an average size of less than 2 micrometers, for inhalation or transdermal 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.
  • 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:
  • 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.
  • 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).
  • NMD number mean diameter
  • VMD volume mean diameter
  • PI polydispersity index
  • 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:
  • the viscosities of the t-butanol-water mixtures were determined experimentally in the 20-30 0 C temperature range.
  • Figure 1 represents the chemical structure of bis-(taurine)-L-glutaminyl-N-palmitoyl-S-[2- (R)-3-dilauroyloxypropyl]-L-cystine (JBT 3002) (C 55 Hi 02 N 4 Oi 4 S 3 Na 2 ).
  • 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 ( 0 C).
  • the scanning rate is of
  • 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 100 0 C 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 ( 0 C).
  • 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 50.27 0 C with a maximum at 39.74 0 C, an onset at 36.25 0 C and a transition enthalpy of 24.27 J/g.
  • the peak around 100 0 C 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.
  • TSA 7ert-butanol 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.
  • 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.
  • MTP-PE muramyl tripeptide phosphatidyl ethanolamine
  • POPC l-palmitoyl-2-oleoyl phosphatidyl choline
  • DOPS 1,2-dioleoyl phosphatidyl serine
  • the preparation was cooled down to room temperature (20 - 25°C), filtered through a standard sterile filter (0.2 ⁇ m) 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 -1O 0 C, warm up within 3h to 20 0 C and dry for 2h.
  • 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.
  • 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 +12O 0 C 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 ⁇ 1.0 0 C, 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
  • POPC l-palmitoyl-2-oleoyl phosphatidyl choline
  • DOPS 1,2- dioleoyl phosphatidyl serine
  • 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.
  • DSC Differential Scanning Calorimetry
  • 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.
  • POPC lipid ratio
  • DOPS 1,2-dioleoyl phosphatidyl serine
  • 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.
  • DSC Differential Scanning Calorimetry
  • 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 50 0 C and stirred until dispersed.
  • 4.0 g of the dispersion were filtered from a syringe directly through a 0.22 ⁇ m 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.
  • DSC Differential Scanning Calorimetry
  • 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 50 0 C and stirred until dispersed.
  • 4.0 g of the dispersion were filtered from a syringe directly through a 0.22 ⁇ m 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.
  • DSC Differential Scanning Calorimetry
  • Table IX DSC of lyophilized powders containing different amount of cholesterol.
  • Example 8 Effect of other phospholipids on lyophilized powder characteristics
  • Table X Composition of lyophilised powders containing additional phospholipids.
  • DSC Differential Scanning Calorimetry
  • 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 ⁇ m 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
  • the liposomes were of multi-lamellar nature as visible by light microscopy in polarized light.
  • Example 9 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 ⁇ m, while allowing air to enter the vial through a hydrophobic filter.
  • the liposomes formed had the following size characteristics:
  • Example 12 Colloidal characteristics of phospholipid blends dispersed in t- butanol-water mixtures
  • 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.
  • colloidal suspensions are significantly more polydispersed in size than in J-butanol-dihydrate.
  • Phospholipid concentration 7 % (weight ratio).
  • 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
  • test solution One hundred microliters of the test solution were added to the wells of a 48-flat-bottom well plate.
  • TNF- ⁇ measurements were performed on macrophage supernatants according to an established protocol "Cytokine (TNF- ⁇ ) Measurement by ELISA", using appropriate dilutions series of the supernatants as required by this protocol. Quantikine human TNF- ⁇ (R&D Systems, Cat n° STAOOC) was used as the reference standard. 50% effect concentrations were calculated according to generally established methods.
  • 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- ⁇ concentration. MTP-PE liposomal formulation lots induced a dose dependent TNF- ⁇ secretion from macrophages.
  • TNF- ⁇ 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 ⁇ g/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 ⁇ m. 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.
  • Example 2 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 ⁇ m before micronization and 4 ⁇ m 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.
  • 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.
  • Example 15 Injectable powder / microsranulate 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).
  • phagocytic cells monocytes, macrophages and dendritic cells
  • innate immune response protection against the exogenous or abnormal antigens
  • adaptive T cell response specific to the lipophilic antigens.
  • Example 2 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.
  • Niro Atomizer Portable spray drier Cold 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 ⁇ m 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 ⁇ g 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.
  • 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.
  • 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.
  • 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 ⁇ m pore size into clean and sterile vials of 2 ml volume. 200 ⁇ l 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 - 24 0 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.

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Abstract

L'invention concerne une poudre homogène comprenant ou constituée de : 0,01-20 % (p/p) d'une ou de plusieurs substances amphiphiles ayant des paramètres de solubilité dd compris entre 15,0 et 23,0 J1/2/cm3/2 et des valeurs de (dp2 + dh2)1/2 comprises entre 6,0 et 13,0 J1/2/cm3/2, 80-99,99 % (p/p) d'une combinaison de lipides constituée de : i) 65-95 % (p:p) de palmitoyloléoylphosphatidylcholine (POPC), ii) 5-35 % (p/p) de dioléoylphosphatidylsérine (DOPS), iii) 0-20 % (p/p) d'autres phospholipides, 0-10 % (p/p) de cholestérol, ladite poudre étant encore caractérisée en ce qu'elle a un volume massique de 4-40 ml/g de lipides mélangés, en ce qu'elle présente sur un tracé de calorimétrie différentielle à balayage (DSC) une seule transition endothermique avec un pic monomodal ayant un maximum à 30-50°C et une enthalpie de transition de 10 à 30 J/g.
EP06762930A 2005-08-02 2006-08-01 Procédé pour la préparation de formulations liposomiques Withdrawn EP1909758A1 (fr)

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JP2013537518A (ja) 2010-07-06 2013-10-03 ノバルティス アーゲー ＲＮＡ送達に有利なｐＫａ値を有する脂質を含むリポソーム
DK2591114T3 (en) 2010-07-06 2016-08-29 Glaxosmithkline Biologicals Sa Immunization of large mammals with low doses of RNA
DK3243526T3 (da) 2010-07-06 2020-02-17 Glaxosmithkline Biologicals Sa Levering af rna til at udløse flere immunsignalveje
BR112013004866A2 (pt) * 2010-08-31 2016-06-07 Novartis Ag lipossomas peguilados para entrega de rna de imunogénio codificado
KR102266691B1 (ko) 2010-10-11 2021-06-23 노파르티스 아게 항원 전달 플랫폼
EP3854413A1 (fr) 2011-07-06 2021-07-28 GlaxoSmithKline Biologicals SA Compositions de combinaisons immunogènes et utilisations de celles-ci
EP3094345B1 (fr) * 2014-01-17 2018-03-14 Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol Immunothérapie à base de liposome
CN116059219A (zh) 2015-07-16 2023-05-05 比奥克斯塞尔医疗股份有限公司 一种使用免疫调节治疗癌症的新颖方法
JP2018530620A (ja) * 2015-10-19 2018-10-18 カディラ・ヘルスケア・リミテッド 同一の分野の発明を含有する新規アジュバント及びワクチン組成物
FR3119325B1 (fr) 2021-01-29 2023-08-11 Renault Jean Yves Compositions liposomales orales

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CA1260393A (fr) * 1984-10-16 1989-09-26 Lajos Tarcsay Liposomes de lipides synthetiques
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