WO1995015746A1 - Systemes d'administration de liposomes - Google Patents

Systemes d'administration de liposomes Download PDF

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Publication number
WO1995015746A1
WO1995015746A1 PCT/GB1994/002702 GB9402702W WO9515746A1 WO 1995015746 A1 WO1995015746 A1 WO 1995015746A1 GB 9402702 W GB9402702 W GB 9402702W WO 9515746 A1 WO9515746 A1 WO 9515746A1
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Prior art keywords
liposomes
molecule
complex
receptor
cyclodextrin
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PCT/GB1994/002702
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English (en)
Inventor
Gregory Gregoriadis
Brenda Mccormack
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The School Of Pharmacy
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Publication date
Priority claimed from GB939325276A external-priority patent/GB9325276D0/en
Priority claimed from GB939325277A external-priority patent/GB9325277D0/en
Application filed by The School Of Pharmacy filed Critical The School Of Pharmacy
Publication of WO1995015746A1 publication Critical patent/WO1995015746A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • Liposomes may be used in a variety of pharmaceutical applications, in particular as delivery systems for drugs and other substances. Drugs and other substances may be entrapped in the liposomes, for instance by dissolution in the aqueous phase of the liposomes or incorporation in the lipid phase (the bilayer) . Delivery of drugs and other substances, for instance intravenously, orally or transdermally, using liposomes can result in various benefits, for instance increased half-life of drugs in blood circulation and targeting of drugs or other substances to specific sites in the body, for instance diseased cells or lymph nodes (particularly useful in the administration of vaccines) . Liposomes are also known to be useful for topical administration of actives (useful for instance in the cosmetics industry) .
  • the solvent contained within the liposomes is water-based. Therefore entrapment of water soluble or hydrophilic substances is not problematic. Such substances may be dissolved in the aqueous phase. Difficulties are however encountered when it is attempted to entrap substances having low water solubility.
  • DMSO is the encapsulated solvent rather than one which is water-based.
  • DMSO is capable of solvating hydrophobic substances. This method is however not useful in practice. Large amounts of solvent are required to solvate the hydrophobic substances (larger amounts than the amount of water required to solvate an equivalent molar amount . of a hydrophilic substance) making the method expensive.
  • the liposomes will be large, which is not preferred.
  • DMSO can be toxic in large amounts; for instance because it interacts with cell membranes.
  • Cyclodextrin molecules have a geometry which may be described as annular or "bucket” shaped.
  • the geometry and electronic structure of a cyclodextrin molecule is such that the interior surface of the "bucket” is hydrophobic and the external surface of the "bucket” is hydrophilic.
  • a cyclodextrin molecule is illustrated schematically in Figure 1, showing the hydrophobic cavity l and the hydrophilic outer surface 3.
  • cyclodextrins are their ability to form inclusion complexes ("host-guest" complexes) with a wide range of substances.
  • host-guest inclusion complexes
  • Molecular encapsulation of guest molecules within host cyclodextrins allows modification of the apparent physical and chemical properties of these guest molecules. In particular solubility properties may be modified.
  • the cyclodextrins are water-soluble. This characteristic derives from the location of all three hydroxyl groups of each successive glucose unit on the rims of the bucket-shaped molecules - the C 6 primary hydroxyls 5 on the narrower side 7 and the C 2 and C 3 secondary hydroxyls 9 occupying the wider side 11. These two hydrophilic planes confer hydrophilicity upon the entire molecule.
  • hydrophobic molecules may be encapsulated by the cyclodextrin molecule.
  • the driving force for formation of such a host-guest complex is displacement of water molecules from the hydrophobic cavity by the more hydrophobic guest molecule to attain a polar-apolar association and decrease of cyclodextrin ring strain, resulting in a more stable lower energy state.
  • cr-, ⁇ -, and 7-cyclodextrins have a "depth" along the curved wall of about 7.8 angstroms.
  • An ⁇ -cyclodextrin has a cavity of diameter about 5.7 angstroms and a total diameter on the wider side of about 13.7 angstroms. These dimensions in a ⁇ -cyclodextrin are around 7.8 angstroms and 15.3 angstroms respectively. In a ⁇ -cyclodextrin they are around 9.5 angstroms and 16.9 angstroms respectively.
  • Cyclodextrins have also been used to improve a variety of other properties of drugs.
  • the encapsulation of active ingredient may protect it from its environment and thus from reactions which may adversely affect its storage stability, such as hydrolysis, oxidation or volatilisation. Encapsulation may also mask unpleasant tastes and reduce local irritancy and haemolysis.
  • Cyclodextrins have been incorporated into vesicles by Bellanger and Perly described in "Amphiphilic Cyclodextrin Derivatives As Potential Vectors For Hydrophobic Drugs", Sixth International Cyclodextrin Symposium, Chicago 1992. Cyclodextrin molecules were covalently attached to phospholipids to give compounds named "lollipops", the lipid parts of which can be incorporated into the lipid bilayer of organised structures such as liposomes, with the cyclodextrin moiety thus attached to the inner or outer surface of the lipid bilayer of such liposomes and extending therefrom.
  • microcapsules containing cyclodextrins in "Microcapsules of drug-cyclodextrin complexes".
  • the preparation was described of microcapsules having a polymeric coating which encapsulates a solution of a 2- hydroxypropy1- ⁇ -cyclodextrin complex ofhydrocortisone.
  • the polymeric coating is dissolved by gastric fluid, allowing release of the cyclodextrin complex.
  • Cyclodextrins and cyclodextrin complexes tend to be eliminated unchanged by the kidneys very rapidly, thus not allowing release of the encapsulated substance into the body. Further it has been found that CD's can be toxic to the kidneys.
  • the molecule of the drug or other substance may be replaced in the cyclodextrin by, for instance, components in the plasma (e.g plasma proteins) gastric contents and other biological fluids. Thus the drug or other substance may be released prematurely.
  • the plasma e.g plasma proteins
  • Cyclodextrins may react with a complex lipophilic component in the body. Cyclodextrins tend to extract components of bio-surfaces they encounter, for example the surfaces of erythrocytes and also lipoproteins. This behaviour also results in the membrane solubilising properties of cyclodextrins, which can be detrimental if large amounts of cyclodextrin are administered.
  • liposomes containing in the aqueous phase inside the liposome at least one complex, wherein the complex comprises a molecule non-covalently bound to a receptor.
  • the complex comprises a molecule non-covalently bound to a receptor.
  • the non-covalent binding of said molecule to said at least one receptor is effective to modify the interactions of said molecule with other components of the liposome, more preferably effective to modify the solubility properties of said molecule and/or the interaction of said molecule with the lipid bilayer.
  • Two embodiments of the invention in particular are important.
  • liposomes containing in the aqueous phase inside the liposome at least one complex, wherein the complex comprises a hydrophobic molecule non-covalently bound to a receptor, and wherein the complex is hydrophilic.
  • liposomes containing in the aqueous phase inside the liposome at least one complex, wherein the complex comprises a molecule having a tendency to escape from liposomes, non-covalently bound to a receptor and wherein the complex has a tendency to escape from liposomes which is lower than that of the molecule.
  • liposomes as a delivery system for drugs or other substances into the body.
  • liposome system of the invention allows the solubilisation of hydrophobic substances in the aqueous phase of liposomes and hence facilitates their encapsulation in said liposomes.
  • benefits obtainable with delivery using liposomes are accessible for hydrophobic substances as well as for hydrophilic substances. These benefits include increase in the half- life of substances in the circulation, compared with the half-life of the cyclodextrin - active substance complexes mentioned above or of the active substances alone, and direction to particular regions of the body.
  • the system of the invention allows the incorporation into liposomes of larger amounts of hydrophobic substances than has previously been possible.
  • the use of the system of the invention allows the inclusion in the aqueous phase of a single liposome drugs which would normally be incompatible in that environment.
  • the drugs may be rendered inactive or reduced in activity towards each other by their complexation with a receptor or receptors.
  • a small molecule is non-covalently bound to a receptor which is not able to escape easily from the liposomes through the lipid bilayer. Thus the molecule is trapped inside the liposome.
  • the liposomes may be unilamellar or multilamellar. They may have mean diameter up to 50 ⁇ m but preferably have a mean diameter of 200nm or less.
  • the liposomes may be produced from liposome forming material, preferably lipid.
  • Lipids used may be one or more of phosphatidylcholine, cholesterol , phosphatidylglycerol , phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, sphingomyelin or derivatives, for instance distearyl, dirmyristoyl and dipal itoyl derivatives, of these lipids, preferably phosphatidylcholine or cholesterol.
  • the percent entrapment of complex into and stability of the resulting liposomes is influenced by the choice of lipid material used to make the liposomes.
  • it is advantageous to include cholesterol in the liquid bilayer of liposomes made from phospholipids having low gel-liquid crystallisation temperature (Tc) such as phosphatidylcholine and dimyristoyl phosphatidylcholine.
  • Tc gel-liquid crystallisation temperature
  • the absence of cholesterol is advantageous in liposomes whose lipid bilayer is made from high Tc phospholipids, such as dipalmitoyl phosphatidylcholine. Both systems can result in greater entrapment values.
  • the systems tend to exhibit reduced loss of receptor without drug from the vesicle. It is believed that the respective presence and absence of cholesterol in these environments gives these results due to promotion of bilayer rigidity.
  • substances which may be complexed with a receptor and subsequently entrapped in the liposomes are pharmaceuticals, vaccines, genetic materials, enzymes, hormones, vitamins, carbohydrates, proteins/peptides, lipids, organic molecules and inorganic molecules or atoms. More specific examples are anti-tumour and anti-microbial agents, enzymes, hormones, vitamins, metal chelators and genetic material, preferably carbohydrates or proteins/peptides. Specific examples include morphine, indomethacin, Naproxen, Ketoprofen, tin etiopurpurin, pilocarpine, hydrocortisone, oestrogen, progesterone, prostaglandins, cholesterol.
  • the complexed and entrapped molecule of the first important embodiment of the invention is hydrophobic and hence has low water solubility.
  • examples of such substances are dehydroepiandrosterone (DHEA) , retinoic acid, retinol, chlorambucil, dexamethasone, indomethacin, /3-Tocopherol, Vitamin E, Vitamin D.
  • DHEA dehydroepiandrosterone
  • retinoic acid retinol
  • chlorambucil dexamethasone
  • indomethacin /3-Tocopherol
  • Vitamin E Vitamin D
  • Vitamin D Vitamin D
  • the second important embodiment of the invention covers the complexation and entrapment of small molecules with a tendency to leak from liposomes. Examples of such substances are melaphalon, vincristine.
  • the liposome systems of the invention are useful for both systemic and topical administration.
  • the receptor used for the first important embodiment of the invention is usefully any substance capable of forming a complex with a hydrophobic molecule in order to modify the apparent solubility properties of the said hydrophobic molecule.
  • Complexation is preferably by engulfment or encapsulation of the hydrophobic molecule. More than one hydrophobic molecule may be bound by one receptor. Conversely more than one receptor may bind one hydrophobic molecule. More than one type of molecule may be bound by a single receptor. Examples of such substances are binding proteins having a hydrophobic pocket into which a small hydrophobic molecule may be received and an external surface which is hydrophilic, hence rendering the binding protein and the complex of the hydrophobic molecule with the binding protein water soluble.
  • a particularly suitable group of substances for use as the receptor are those which have an annular molecular geometry.
  • the molecules are "bucket-like", having a cavity which can receive molecules of suitable size and character.
  • An example of a substance of this type is cyclopolygalacturonic acid (CPGA) .
  • CPGA cyclopolygalacturonic acid
  • the CPGA molecule has a ring structure which allows engulfment of lipids or lipid-like materials.
  • Further substances of this type are calixarene based substances.
  • Calixarenes have a "bucket- like" geometry. They are based on phenol molecules joined in a ring by alkyl groups. The benzene rings from the walls of the "bucket”.
  • a further suitable class of receptors of this type is the class of cyclodextrins and cyclodextrin derivatives. This class of material is the most preferred for use in the first important embodiment of the invention.
  • the receptor for use in the second important embodiment of the invention is usually any substance capable of forming a complex with a small molecule with a natural tendency to escape from liposomes said complexation being effective to modify the apparent leakage properties of the molecule.
  • the receptor itself should have a low tendency to escape from liposomes, hence the receptor-drug complex will have a low tendency to escape.
  • Binding proteins having a hydrophilic pocket can form complexes with small hydrophilic molecules. Binding proteins have a low tendency to leak from liposomes due to their large molecular weight. Thus the complex itself remains within the liposome.
  • the receptor in this second important embodiment of the invention also it is preferred to use as the receptor a substance having annular molecular geometry, in particular a cyclodextrin or cyclodextrin derivative.
  • the system of the invention pre erably includes as the receptor a cyclodextrin or cyclodextrin derivative. This system has advantages over previously tested delivery systems based on administration of cyclodextrin-drug complexes alone.
  • Cyclodextrins like sugars in general, do not tend to leak out through the lipid bilayer, even when attempts have not been made to decrease the permeability of the bilayer. Thus small molecules with a tendency to leak from the liposomes can be rendered "non-leaky" by inclusion in a cyclodextrin host-guest complex. Natural cyclodextrins may be used, that is ⁇ -, ⁇ - or 7- cyclodextrin. ⁇ -cyclodextrin is the most readily available and lowest-priced of these.
  • Natural cyclodextrins are produced from starch by the action of cyclodextrin glycosyltransferase (CTG) , an amylase-type enzyme usually obtained from cultures of the micro-organism Bacillus Macerans. Natural cyclodextrins are commercially available, for instance from American Maize-Products Company. Other methods of encapsulation are applicable, for instance the microfluidation method, which would not normally be appropriate for the encapsulation of non- ccmplexed water-insoluble substances; detergent dialysis may also be used, as may methods involving solid lipids. Cyclodextrins may be derivatised in various ways.
  • cyclodextrins may be chemically substituted to give methylated cyclodextrins or hydroxypropyl cyclodextrins, which have a much greater water solubility than their corresponding parent cyclodextrins.
  • Methods of substituting cyclodextrins include monosubstitution on primary-hydroxyl side, monosubstitution on secondary- hydroxyl side, disubstitution on adjacent glucopyranose units, disubstitution on alternate glucopyranose units, disubstitution on diametrically opposite glucopyranose units, appending, capping, double capping, duplexing with single bridge and duplexing with double bridge.
  • Derivatisation may be carried out by conventional chemical methods known in the art.
  • hydroxypropyl derivatives are less toxic than their natural counterparts and that toxicity of cyclodextrins increases in the order 7 ⁇ ⁇ ⁇ .
  • haemolytic activities of cyclodextrin sulphates and 2-hydroxypropyl- ⁇ -cyclodextrin are lower than those of their parent cyclodextrins.
  • Derivatives which can be used include methyl, ethyl, pentyl, hydroxypropyl, 2-hydroxypropyl, hydroxyethyl, amino, deoxy, glucosyl, maltosyl, heptakis-2,6-dimethyl, O- carboxymethyl-O-ethyl and sulphate derivatives.
  • Derivatisation can be very useful in modifying the properties of cyclodextrins, for instance their solubility, toxicity and inclusion properties.
  • Soluble polymers and dimers of cyclodextrins or their derivatives may also be used as the receptor. Any of the above mentioned cyclodextrins may be used in dimerised or polymerised form provided such dimerisation or polymerisation is possible. Polymers of j8-cyclodextrin are commercially available, for instance from Cyclolab (Budapest, Hungary) .
  • Molecular weight may vary from for instance 1,900 to 13,000, generally from around 4,000 to around 9,100.
  • the number of cyclodextrin units present in the polymer may be any number from 2 upwards, and is usually not more than 10 or 15 units. Most usually the cyclodextrin polymers contain from 3 to 8 units on average per polymer molecule.
  • Commercially available 3-cyclodextrin polymers have average molecular weights of for instance 4,000 to 4,500 and around 8,700.
  • Cyclodextrin polymers may be homopolymers of one type of cyclodextrin monomer. Alternatively they may be copolymers or terpolymers of more than one type of cyclodextrin monomer. Alternatively or additionally further comonomers may be present which are not cyclodextrins.
  • the polymers may be graft copolymers of cyclodextrin onto a polymeric backbone containing suitable groups.
  • This backbone may be for instance polyvinylimidazole.
  • random copolymers may be produced, for instance with epichlorhydrin monomer.
  • epichlorhydrin or other bifunctional agents such as diepoxide, diester and diisocyanate as comonomer can give cross-linking of the cyclodextrin polymer.
  • Random copolymerisation and graft copolymerisation can be promoted by the introduction of polymerisable groups into the cyclodextrin or cyclodextrin derivative. Examples of such groups include the (meth)acryloyl group.
  • polymers Conventional methods of polymerisation may be used for production of homopolymers or copolymers. These include reverse phase emulsion polymerisation and inverse suspension bead polymerisation. Examples of polymers which are useful include those prepared by reacting a mono-substituted 6-o-(3-chloro-2- hydroxypropyl) 3-cyclodextrin derivative with the amine groups of polyvinylimidazole to give a graft copolymer.
  • a further exemplary polymer may be produced by introducing polymerisable methacryloyl groups into ⁇ -, ⁇ - and 7- cyclodextrins or their hydroxyalkylated derivatives and carrying out radicalic inverse suspension polymerisation of the water-soluble monomers with or without comonomers to give a water-swelling bead polymer of particle size 5-100 ⁇ m.
  • inclusion complexes There are various methods known for the production of inclusion complexes.
  • one method of inclusion by slurry mixing the appropriate amount of guest compound (alone or dissolved in an appropriate solvent) is added to a slurry of cyclodextrin prepared with 0.3 to 3.0 (generally l.o to 2.0) times its own weight of water and mixed thoroughly for 0.5 hour to several hours using homogeniser, mortar etc.
  • the viscosity may increase giving a paste.
  • the complex is appropriately washed and dried.
  • Complexation may also be effected by additions of water-insoluble active, dry or dissolved in a non-aqueous solvent, to an aqueous solution of cyclodextrin (active usually in excess although not always) and mixing, for instance by sonication or stirring. Complexation may be evidenced by the dissolution of the water-insoluble active substance.
  • the active-cd complex may be separated from uncomplexed components. This may be achieved by filtration or centrifugation, for instance, if the complex is insoluble. Filtration may be by any suitable method, for instance through packed glass wool. For the water-soluble complexes of the present invention chromatographic separation may be used, for instance using a Sephadex G 10 column.
  • Complexes of cyclodextrins with hydrophilic substances may be formed by a method of saturation in aqueous solution, in which a water-soluble compound is added directly to a saturated aqueous solution of cyclodextrin and the mixture is slowly agitated for from 0.5 hour to several hours to form the complex.
  • the complex precipitates at room temperature or on cooling and is isolated by filtration and drying.
  • This method may be used for water-insoluble compounds, which are first dissolved in the minimum quantity of a suitable solvent, for instance acetone, before addition to the cyclodextrin solution.
  • the receptor:drug molar ratio is usually 1:1 but complexes may be formed with a higher or lower proportion of receptor.
  • the stability of the receptor-drug complex is very important. A balance is required between lability, replacement of the active substance by another molecule in vivo and hence premature release of the administered substance and very high stability leading to retarded or incomplete release of the administered substance in vivo. Stability constants within the desired range may be found by a skilled person. If the receptor is a cyclodextrin, stability constants may be modified by selective derivatisation of natural cyclodextrins.
  • step (b) subjecting a solution of the complex formed in step (a) to gel permeation chromatography to separate the complex from non-complexed guest molecules.
  • This process is especially useful in the preparation of complexes which are suitable for incorporation into the liposomes of the invention.
  • step a) it is preferred for the product solution from step a) to be passed straight to step b) , although in some circumstances it is preferred for the product solution to be subjected to intermediate purification or recovery steps. Furthermore it is preferred for the product solution to be transferred to the step b) without further additions, although in some cases it may be desirable for additional solvent or other diluent to be added.
  • the encapsulation of the active ingredient in the first step may be achieved by dissolving the receptor molecule into a solvent and then adding the guest molecule, either as a solid or slurry or as a solution in the same or another solvent. The mixture is then mixed thoroughly for 0.5 hour to several hours until the complex formation has taken place.
  • the separation step, step b) is carried out using the usual apparatus and arrangement of gel.
  • the gel is supported in a column.
  • a particularly suitable column is a Sephadex G column.
  • the receptor:guest molar ratio is usually 1:1 but complexes may be formed with a higher or lower proportion of receptor molecule.
  • the receptor molecule may be any of the receptors useful in the liposomes of the invention, such as cyclodextrin-based compounds. These include natural cyclodextrin monomer, derivatised cyclodextrin monomer and cyclodextrin-containing polymer.
  • the guest molecule may be any of the active molecules useful in the liposomes of the invention.
  • the guest molecule may be a hydrophobic molecule. It may be a molecule which has a tendency to escape from liposomes when non-complexed.
  • the invention will be illustrated with reference to specific examples. These illustrate entrapment of the hydrophobic substances when complexed with cyclodextrin and the stability of the resulting liposomes in blood plasma.
  • Entrapment of drv HPgcd inclusion complexes into liposomes Example 1 Entrapment of dehydroepiandrosterone (DHEA) hydroxypropyl-j8-cyclodextrin inclusion complex into the aqueous phase of liposomes.
  • DHEA dehydroepiandrosterone
  • the thin lipid film was suspended in 2ml water at a temperature (Ta) above the gel-liquid crystalline transition temperature (Tc) of the phospholipid.
  • Ta gel-liquid crystalline transition temperature
  • Tc gel-liquid crystalline transition temperature
  • the suspension was then probe sonicated at Ta to produce small unilamellar vesicles (SUV) . These were allowed to stand for 60min at Ta and then mixed with 0.05ml of the HP-jffcd-DHEA complex.
  • the mixture was diluted to 3 or 10ml with H 2 0 and freeze-dried overnight. To the freeze-dried material 0.1ml of H 2 0 was added at Ta and the sample swirled vigorously and allowed to stand for 30min at Ta.
  • DHEA-HP-j8cd complex was entrapped in DRV liposomes composed of the lipids shown (phospholipid to cholesterol and phospholipid to phosphatidic acid molar ratios were 1:1 and 1:0.1 respectively).
  • 3 H: C ratios approximating 1.0 signify similar entrapment values for the two isotopes, i.e. entrapment of the intact inclusion complex. Note poor entrapment values and/or 3H:1-4C ratios when volume of freeze-dried liposomes is low.
  • Example 2 Entrapment of carboxyfluorescein (CF)-HP- ⁇ cd complex into the aqueous phase of liposomes. 0.2ml of 0.2M CF was mixed with 0.05ml of HP-3cd (22.5mg) into which 1.15 x 10 5 dpm of 4 C-labelled HP-/3cd had been previously added.
  • CF carboxyfluorescein
  • Example 3 Entrapment of retinoic acid (RA)-HP- / 8cd inclusion complex into the aqueous phase of liposomes. lOmg retinoic acid were dissolved in 2ml CHC1 3 into which 6.3 x 10 6 dpm of 3H-labelled RA (3H-RA) were added.
  • the dry RA was dissolved in 2ml solution of HP-/3cd (400mg) containing 6.5 x 10 5 dpm of u C-HP-/3cd by stirring at 37°C for 3 days.
  • the solution formed (milky suspension) was centrifuged at 100,000g for 1 h.
  • Most of the inclusion complex of RA-HP- jScd was recovered in the supernatant. Formation of the inclusion complex was verified by molecular sieve chromatography on a Sephadex G10 column: the fraction containing U C-HP-j8cd (peak at fraction 10) also contained most of the 3H-RA.
  • Example 4 Entrapment of retinol (R)-HP-3cd inclusion complex into the aqueous phase of liposomes.
  • lO g of retinol were dissolved in 2ml CHC1 3 into which 7.2 x 10° dpm of 3 H-labelled R( 3 H-R) were added.
  • the dry R was dissolved in 2ml of HP-3cd (lOOmg) containing 6.5 x 10 5 dpm of u C-HP-/3cd by stirring at 37°C for 2 days.
  • Verification of inclusion complex formation in the clear solution was carried out by molecular sieve chromatography using Sephadex G10: most of the inclusion complex of H-R- C-HP-/_?cd was recovered in the fraction also containing the C-HP-3cd.
  • 0.5ml of the R-HP-3cd inclusion complex (2.5mg of R and 25mg of HP-/3cd) was used for entrapment into DRV as in Example 1 (for conditions see Table 4) .
  • Entrapment of R-HP-/3cd inclusion complex was estimated on the basis of H-R and 14 C-HP-/Scd radioactivity (Table 4) .
  • Example 5 Entrapment of DHEA 3-cyclodextrin polymer inclusion complex into the aqueous phase of liposomes.
  • the solutions formed were filtered through packed glass wool to remove non-solubilised matter. Final molar ratios were not estimated because radio-labelled polymers were unavailable.
  • H.R/ HP-3-cd inclusion complex was entrapped in liposomes of the composition shown. Note t low entrapment values obtained with PC/CHOL liposomes.
  • 0.3ml of liposome-entrapped RA-HP-/Scd inclusion complex, radiolabelled with 3 H(RA) and U C-(HP-Scd) was mixed with 0.6ml rat blood plasma or 0.6ml PBS and incubated at 37°C for time intervals.
  • 0.4ml and 0.5ml samples were taken at 2 min and 60 min respectively from the incubated samples and tested for inclusion complex release by centrifugation at 100,000g for 25 min of the sample diluted to 5ml with water.
  • DRV liposomes containing RA/HP-3-cd complex were incubated at 37°C in the presence of PBS (control) or rat blood plasma. Values denote released 3 H and 14 C as % of amounts incubated. Note the small additional effect of plasma when compared to that of PBS. Results indicate that, in terms of RA release, DSPC/CHOL liposomes are less stable than those made of DSPC.
  • DRV liposomes containing R/HP/S-cd complex were incubated at 37°C in the presence of PBS or rat blood plasma. Values denote released 3H and 14C as % of amounts incubated. Note the small additional effect of plasma when compared to that of PBS. Results indicate that, in terms of R release, DSPC/CHOL liposomes are less stable than those made of DSPC. For other comments see Example 6. These examples indicate that hydrophobic or water- insoluble substances may be entrapped in the aqueous phase of liposomes by means of the formation of a cyclodextrin inclusion complex. The resulting liposomes are stable in blood plasma.
  • 3 H-DXM/ 14 C-HP-/3cd complexes were prepared using methods as described above.
  • the complexes were entrapped in DRV liposomes comprised of DSPC only, using methods as described above.
  • the liposome preparations were incubated in the presence of PBS or rat plasma at 37°C for 2 and 60 minutes. At the end of the incubation period samples were centrifuged to sediment liposome. Released radioactivity ( 3 H and H C) was measured in the supernatant.
  • Example 9 Behaviour of drug-cd complexes in vivo Inclusion complexes were prepared of DHEA, retinol (R) and dexamethasone (DXM) , each labelled with H, were prepared with u C-(HP-Scd) as described above.
  • Some of each complex was entrapped in DRV liposomes as described above. The liposomes were made of DSPC only.
  • Rats in groups of four were injected intravenously with 1.0 ml of PBS containing free or liposomal complex.
  • the dose of free DHEA/HP-/3cd comprised 5 mg DHEA and 2 mg HP-/Scd.
  • the dose of liposomal DHEA/HP-/3cd comprised 2 mg DHEA and 0.45 mg HP-Scd.
  • the dose of free R/HP-/3cd comprised 0.2 mg R and 4.5 mg P- ⁇ cd.
  • 8cd complex comprised 0.05 mg R and 0.6 mg HP-jScd.
  • Each dose of free DXM/HP-Scd complex comprised 0.2 mg DXM and 0.6 mg HP-jScd.
  • Each dose of liposomal DXM/HP-/3cd complex comprised 0.1 mg DXM and 0.2 mg HP-/3cd. Some animals were killed at 30 minutes after injection. Others were killed at 24 hours after injection. Content of drug ( H content) and KP- ⁇ cd ( C content) were measured in the blood plasma, liver, spleen and kidneys of the animals.
  • Liposomal R/HP ⁇ cd complex 1.3 ⁇ 0.2 1.0 ⁇ 1.0 1.3 0.9 ⁇ 0.1 0.1 ⁇ 0.0 9.0
  • Liposomal DXM/HP ⁇ cd 1.0 ⁇ 0. 1 0.25 ⁇ 0.0 4.0 0.2 ⁇ 0.1 0. 1 ⁇ 0.0 2.0 complex
  • Free DHEA/HP ⁇ cd complex 19.4 ⁇ 2.2 1.0 ⁇ 0.1 19.4 0.9 ⁇ 0.2 0.3 ⁇ 0.1 3.0
  • Free DHEA/HP ⁇ cd complex 0.1 ⁇ 0.0 0.1 ⁇ 0.0 1.0 0.0 0.0 1.0
  • Free R/HP ⁇ cd complex 1.2 ⁇ 0.6 0.3 ⁇ 0.1 4.0 0.2 ⁇ 0.1 0.1 ⁇ 0.0 2.0
  • Free DXM/HP ⁇ cd complex 0.5 ⁇ 0.2 0.2 ⁇ 0.0 2.5 0.1 ⁇ 0.0 0.1 ⁇ 0.0 1.0
  • Example 10 Recovery of drug/HP/Scd inclusion complexes in urine of rats Rats were injected as in Example 8. The animals which were killed at 24 hours were kept individually in metabolic cages with facility for 24 hour urine collection. Levels of 3 H (drug) and U C (HP-?cd) were measured. In the urine collected over the 24 hour period. Results are shown in Table 11 below.
  • Liposomal DHEA/HPjScd complex 25.1 ⁇ 2.1 6.4 ⁇ 1.2 3.9
  • Liposomal R/HPj8cd complex 26.1 ⁇ 5.9 20.8 ⁇ 0.0 1.25
  • Examples 9 and 10 show that liposome-entrapped inclusion complexes have a reduced tendency to be excreted rapidly in the urine compared with free inclusion complexes and have a greater tendency to travel to the tissues.
  • Table 8 shows that the non-entrapped complexes have a greater tendency to remain in the circulation than the liposome-entrapped complexes.
  • the results shown in Tables 9 and 10 illustrate that the liposomal complexes when removed from the circulation have a tendency to travel to the tissues, where the drugs will of course be required. This is illustrated by their presence in the liver and spleen in greater amounts at 30 minutes than the free inclusion complexes.

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Abstract

L'invention se rapporte à des liposomes qui contiennent dans leur phase aqueuse au moins un complexe. Le complexe comprend au moins une molécule liée par covalence à au moins un récepteur. De préférence, cette liaison est efficace pour modifier les interactions de cette molécule avec d'autres composants du liposome, par exemple ses propriétés de solubilité. Ce système peut être utilisé pour incorporer dans des liposomes des molécules hydrophobes, insolubles dans l'eau et/ou des molécules qui tendent à s'échapper des liposomes lorsqu'elles ne sont pas liées à un récepteur. La molécule liée peut être un agent pharmaceutique, actif, et dans ce cas les liposomes peuvent être utilisés dans un système d'administration de médicaments.
PCT/GB1994/002702 1993-12-10 1994-12-09 Systemes d'administration de liposomes WO1995015746A1 (fr)

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GB9325277.3 1993-12-10
GB9325276.5 1993-12-10
GB939325276A GB9325276D0 (en) 1993-12-10 1993-12-10 Separating complexes
GB939325277A GB9325277D0 (en) 1993-12-10 1993-12-10 Liposome delivery system

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0751150A1 (fr) * 1995-06-29 1997-01-02 Commissariat A L'energie Atomique Dérivés de cyclodextrines, leur préparation et leur utilisation pour incorporer des molecules hydrophobes dans des systèmes de tensioactifs organisés
WO1997013499A1 (fr) * 1995-10-11 1997-04-17 The University Of British Columbia Formulations de liposomes a base de mitoxantrone
WO2000037109A2 (fr) * 1998-12-18 2000-06-29 Euphar Group S.R.L. Clathrates de dehydroepiandrosterone et compositions pharmaceutiques correspondantes
FR2827765A1 (fr) * 2001-07-27 2003-01-31 Oreal Composition a base de vesicules lamellaires lipidiques incorporant au moins un compose a base de dhea
EP1543841A1 (fr) * 2002-08-15 2005-06-22 Yunqing Liu Formulation nanopharmaceutique et son procede de preparation
WO2006036484A2 (fr) * 2004-09-24 2006-04-06 Rxdino, Llc Traitement de dermatite au moyen de combinaisons de deshydroepiandrosterone-glucocorticoide
WO2006071659A1 (fr) * 2004-12-29 2006-07-06 Trustees Of Boston University Delivrance d'antagonistes h2
WO2013155487A1 (fr) * 2012-04-12 2013-10-17 Yale University Véhicules pour l'administration contrôlée de différents agents pharmaceutiques
JP2017513938A (ja) * 2014-04-10 2017-06-01 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブライ ユニバーシティー オブ エルサレム リミテッドＹｉｓｓｕｍ Ｒｅｓｅａｒｃｈ Ｄｅｖｅｌｏｐｍｅｎｔ Ｃｏｍｐａｎｙ Ｏｆ Ｔｈｅ Ｈｅｂｒｅｗ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｊｅｒｕｓａｌｅｍ Ｌｔｄ． リポソームムピロシン
US9884026B2 (en) 2013-11-01 2018-02-06 Yale University Modular particles for immunotherapy

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EP0261719A2 (fr) * 1986-09-23 1988-03-30 Akzo N.V. Complexes de cyclodextrine thermochemiluminescents
JPH04351950A (ja) * 1991-05-30 1992-12-07 Mitsubishi Electric Corp 臭気感応材料
WO1993011757A1 (fr) * 1991-12-10 1993-06-24 Orion-Yhtymä Oy Compositions de medicaments pour usage parenteral
WO1994023697A1 (fr) * 1993-04-22 1994-10-27 Depotech Corporation Liposomes de cyclodextrine encapsulant des composes pharmacologiques et leurs procedes d'utilisation

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EP0261719A2 (fr) * 1986-09-23 1988-03-30 Akzo N.V. Complexes de cyclodextrine thermochemiluminescents
JPH04351950A (ja) * 1991-05-30 1992-12-07 Mitsubishi Electric Corp 臭気感応材料
WO1993011757A1 (fr) * 1991-12-10 1993-06-24 Orion-Yhtymä Oy Compositions de medicaments pour usage parenteral
WO1994023697A1 (fr) * 1993-04-22 1994-10-27 Depotech Corporation Liposomes de cyclodextrine encapsulant des composes pharmacologiques et leurs procedes d'utilisation

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DATABASE WPI Week 0393, Derwent World Patents Index; AN 93-023654 *

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EP0751150A1 (fr) * 1995-06-29 1997-01-02 Commissariat A L'energie Atomique Dérivés de cyclodextrines, leur préparation et leur utilisation pour incorporer des molecules hydrophobes dans des systèmes de tensioactifs organisés
FR2736056A1 (fr) * 1995-06-29 1997-01-03 Commissariat Energie Atomique Derives de cyclodextrines, leur preparation et leur utilisation pour incorporer des molecules hydrophobes dans des systemes de tensioactifs organises
US5821349A (en) * 1995-06-29 1998-10-13 Commissariat A L'energie Atomique Derivatives of cyclodextrins, their preparation and their use for incorporating hydrophobic molecules into organized surfactant systems
WO1997013499A1 (fr) * 1995-10-11 1997-04-17 The University Of British Columbia Formulations de liposomes a base de mitoxantrone
US5858397A (en) * 1995-10-11 1999-01-12 University Of British Columbia Liposomal formulations of mitoxantrone
WO2000037109A3 (fr) * 1998-12-18 2000-09-14 Euphar Group Srl Clathrates de dehydroepiandrosterone et compositions pharmaceutiques correspondantes
JP2002532565A (ja) * 1998-12-18 2002-10-02 ユーファル グループ ソシエタ ア レスポンサビリタ リミタータ デヒドロエピアンドロステロンの包接化合物及び対応する薬学的組成物
EA003304B1 (ru) * 1998-12-18 2003-04-24 Эуфар Груп С.Р.Л. Клатраты дегидроэпиандростерона и соответствующие фармацевтические композиции
WO2000037109A2 (fr) * 1998-12-18 2000-06-29 Euphar Group S.R.L. Clathrates de dehydroepiandrosterone et compositions pharmaceutiques correspondantes
FR2827765A1 (fr) * 2001-07-27 2003-01-31 Oreal Composition a base de vesicules lamellaires lipidiques incorporant au moins un compose a base de dhea
WO2003011245A1 (fr) * 2001-07-27 2003-02-13 L'oreal Composition a base de vesicules lamellaires lipidiques incorporant au moins un compose a base de dhea
EP1543841A4 (fr) * 2002-08-15 2011-03-16 Yunqing Liu Formulation nanopharmaceutique et son procede de preparation
EP1543841A1 (fr) * 2002-08-15 2005-06-22 Yunqing Liu Formulation nanopharmaceutique et son procede de preparation
WO2006036484A2 (fr) * 2004-09-24 2006-04-06 Rxdino, Llc Traitement de dermatite au moyen de combinaisons de deshydroepiandrosterone-glucocorticoide
WO2006036484A3 (fr) * 2004-09-24 2006-06-01 Rxdino Llc Traitement de dermatite au moyen de combinaisons de deshydroepiandrosterone-glucocorticoide
WO2006071659A1 (fr) * 2004-12-29 2006-07-06 Trustees Of Boston University Delivrance d'antagonistes h2
US10709664B2 (en) 2012-04-12 2020-07-14 Yale University Nanolipogel comprising a polymeric matrix and a lipid shell
US9603800B2 (en) 2012-04-12 2017-03-28 Yale University Methods of treating inflammatory and autoimmune diseases and disorders using nanolipogels
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US10195144B2 (en) 2012-04-12 2019-02-05 Yale University Methods of treating inflammatory and autoimmune diseases and disorders
EP3520780A1 (fr) * 2012-04-12 2019-08-07 Yale University Véhicules pour l'administration contrôlée d'agents pharmaceutiques différents
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WO2013155487A1 (fr) * 2012-04-12 2013-10-17 Yale University Véhicules pour l'administration contrôlée de différents agents pharmaceutiques
US11173119B2 (en) 2012-04-12 2021-11-16 Yale University Nanolipogel vehicles for controlled delivery of different pharmaceutical agents
EP3939572A1 (fr) * 2012-04-12 2022-01-19 Yale University, Inc. Véhicules pour l'administration contrôlée d'agents pharmaceutiques différents
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JP2017513938A (ja) * 2014-04-10 2017-06-01 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブライ ユニバーシティー オブ エルサレム リミテッドＹｉｓｓｕｍ Ｒｅｓｅａｒｃｈ Ｄｅｖｅｌｏｐｍｅｎｔ Ｃｏｍｐａｎｙ Ｏｆ Ｔｈｅ Ｈｅｂｒｅｗ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｊｅｒｕｓａｌｅｍ Ｌｔｄ． リポソームムピロシン

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