WO2008030807A2 - Phosphatidylethanolamine derivative and liposome containing the same - Google Patents

Phosphatidylethanolamine derivative and liposome containing the same Download PDF

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Publication number
WO2008030807A2
WO2008030807A2 PCT/US2007/077537 US2007077537W WO2008030807A2 WO 2008030807 A2 WO2008030807 A2 WO 2008030807A2 US 2007077537 W US2007077537 W US 2007077537W WO 2008030807 A2 WO2008030807 A2 WO 2008030807A2
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liposome
acid
phosphatidylethanolamine
lipid composition
group
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PCT/US2007/077537
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French (fr)
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WO2008030807A3 (en
Inventor
Kazushi Okada
Naoyuki Morisaki
Yusuke Miyajima
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Mebiopharm Co., Ltd.
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Publication of WO2008030807A2 publication Critical patent/WO2008030807A2/en
Publication of WO2008030807A3 publication Critical patent/WO2008030807A3/en

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    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • the present invention relates to a novel phosphatidylethanolamine derivative and a liposome containing the same.
  • initial liposomes make use of naturally occurring cell membrane phospholipids such as egg-yolk phospholipids and soybean phospholipids which upon intravenous administration, are easily incorporated into the reticuloendothelial system such as the liver, spleen and thus there is a problem of low retention in blood.
  • naturally occurring cell membrane phospholipids such as egg-yolk phospholipids and soybean phospholipids which upon intravenous administration, are easily incorporated into the reticuloendothelial system such as the liver, spleen and thus there is a problem of low retention in blood.
  • liposomes (PEG liposomes) wherein synthetic phospholipids whose lipid moiety contains saturated bonds only are used as a constituent of the liposome membrane, and further, the surface of which is modified with polyethylene glycol have been developed and they have been gaining a growing attention as epoch-making liposomes which are highly retainable in blood without being incorporated into the cellular endothelial system.
  • a strong effort as to retain the liposomes in blood has created a problem of side effects such as the hand foot syndrome, that is a side effect brought by Doxil (liposome preparation of doxorubicin) affecting the peripheral system (as described in a package leaflet of Doxil).
  • Doxil liposome preparation of doxorubicin
  • Patent Document 1 A synthetic phospholipid compound having a dicarboxylic acid bound to phosphatidylethanolamine, which is useful for delivery of a pharmaceutical preparation, has been reported (Patent Document 1), but the compound described therein substantially has a polyalkylene glycol moiety, and the problem of the above side effects is a concern.
  • the object of the present invention is to provide a liposome preparation having high efficiency of delivery of a drug into a target cell which exhibits such an appropriate retention in blood as to allow the pharmaceutical preparation to be incorporated into a target cell but not such a long time as to induce its side effects on normal cells, as well as a novel synthetic phospholipid useful in preparation of the liposome preparation.
  • the present invention provides a phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof:
  • the present invention also provides a lipid composition containing the phosphatidylethanolamine derivative (1) described above and other phospholipid.
  • Liposomes using the novel phospholipid of the present invention contain a pharmaceutical preparation such as an anticancer agent, have high storage stability, are delivered highly efficiently into a target cell upon administration, and are retained in blood for an appropriate time to allow the pharmaceutical preparation to be incorporated into a target cell but not retained in blood for such a long time as to induce their side effects on normal cells.
  • a pharmaceutical preparation such as an anticancer agent
  • Fig. 1 shows a 1 H-NMR chart of the phospholipid obtained in Example 1.
  • the alkyl group represented by R 1 in the general formula (1) includes Cl to ClO alkyl groups, and specific examples thereof include a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, t-butyl group, n-pentyl group, n-hexyl group and the like.
  • the cycloalkyl group includes C3 to ClO cycloalkyl groups, and specific examples thereof include a cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group and the like.
  • the aryl group includes C6 to Cl 5 aryl groups, and specific examples thereof include a phenyl group, tolyl group, xylyl group, 1-naphtyl group, 2-naphtyl group and the like.
  • R 1 is preferably an alkyl group or an aryl group, particularly preferably an alkyl group.
  • X is preferably -O- or -NH-, particularly preferably -O-.
  • the acyl group represented by R 2 and R 3 is preferably an acyl group derived from a saturated or unsaturated fatty acid, more preferably a C 12 to C22 acyl group derived from a fatty acid, more preferably a C14 to C20 acyl group derived from a fatty acid.
  • fatty acids from which these acyl groups are derived include linear saturated carboxylic acids such as lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, tuberculostearic acid, arachidic acid, heneicosanoic acid and behenic acid, as well as linear unsaturated carboxylic acids such as 2-lauroleic acid, linderic acid, Tohaku acid, 5-lauroleic acid, 11-lauroleic acid, tsuzuic acid, 5-miristoleic acid, myristoleic acid, 2-palmitoleic acid, 7-palmitoleic acid, cis-9-palmitoleic acid, trans-9-palmitoleic acid, petroselinic acid, petroselidinic acid, oleic acid, elaidic acid, asclepinic acid, vaccenic acid, gondoic acid, trans-gondoic acid,
  • a salt of the phosphatidylethanolamine derivative (1) includes alkali metal salts such as sodium salt, potassium salt and the like, alkaline earth metal salts such as calcium salt, magnesium salt and the like, amine salts, ammonium salts and the like.
  • the phosphatidylethanolamine derivative (1) of the present invention can be produced for example by the following methods (a) to (c):
  • Y represents a hydrogen atom or a metal atom
  • R 1 , R 2 , R 3 , X and m have the same meanings as defined above.
  • a phosphatidylethanolamine dicarboxylic acid derivative represented by the general formula (3) is reacted with a nucleophilic species (4) to give the present compound (1).
  • the phosphatidylethanolamine dicarboxylic acid derivative (3) can be produced by a method described in USP 4,534,899 or a modification thereto.
  • Y in the nucleophilic species (4) includes a hydrogen atom, a metal (MgBr and the like) constituting a Grignard reagent, a metal (CuLi and the like) constituting a Gillman reagent.
  • the compound represented by R 1 XH includes saturated aliphatic hydrocarbon alcohols such as methanol, ethanol; saturated alicyclic hydrocarbon alcohols such as cyclohexanol; aromatic hydrocarbon alcohols such as phenol; saturated aliphatic hydrocarbon amines such as methylamine, ethylamine; saturated alicyclic hydrocarbon amines such as cyclohexylamine; aromatic hydrocarbon amines such as aniline; saturated aliphatic hydrocarbon thiols such as methyl mercaptan; saturated alicyclic hydrocarbon thiols such as cyclohexyl mercaptan; and aromatic hydrocarbon thiols such as thiophenol.
  • saturated aliphatic hydrocarbon alcohols such as methanol, ethanol
  • saturated alicyclic hydrocarbon alcohols such as cyclohexanol
  • aromatic hydrocarbon alcohols such as phenol
  • saturated aliphatic hydrocarbon amines such as methylamine, ethylamine
  • the phosphatidylethanolamine derivative (1) of the invention can be obtained by reacting 50 to 50,000 equivalents, preferably 1,000 to 5,000 equivalents, of the nucleophilic species (4) with the carboxylic acid compound of the formula (3) in the presence of an organic solvent such as chloroform, methylene chloride or the like and a catalyst such as triethyl amine, potassium carbonate, sulfuric acid or the like, at a temperature not higher than the boiling point of the nucleophilic species (4).
  • the amount of the organic solvent used is preferably about half (volume ratio) of the nucleophilic species (4), and the amount of the catalyst added is preferably 1 to 10 equivalents relative to the carboxylic acid compound (3).
  • the phosphatidylethanolamine derivative (1) can be produced by converting the carboxylic acid compound of the formula (3) into the corresponding halide by a halogenating agent such as thionyl chloride, phosphoryl chloride or the like and then reacting it with the nucleophilic species (4). Specifically, the carboxylic acid compound of the formula (3) is reacted with 2 to 5 equivalents of the halogenating agent in the presence of an organic solvent such as chloroform, methylene chloride or the like, to give a halide of the carboxylic acid compound.
  • the amount of the organic solvent used is preferably 1,000 to 5,000 equivalents relative to the carboxylic acid compound of the formula (3).
  • this halide is reacted with the nucleophilic species (4).
  • the halide is reacted with 1 to 5 equivalents, preferably 1.5 to 2 equivalents, of the nucleophilic species (4) at 0°C to room temperature in the presence of an organic solvent such as chloroform, methylene chloride or the like and a catalyst such as triethylamine, potassium carbonate or the like, whereby the phosphatidylethanolamine derivative (1) of the present invention can be obtained.
  • the amount of the organic solvent used is preferably 1,000 to 5,000 equivalents relative to the carboxylic acid compound of the formula (3), and the amount of the catalyst added is preferably 1 to 5 equivalents relative to the nucleophilic species (4).
  • the present compound (1) can be obtained by reacting the carboxylic acid compound (6) or its anhydride with the phosphatidylethanolamine (5).
  • the carboxylic acid compound (6) is a carboxylic acid anhydride
  • the carboxylic acid compound (6) anhydride is reacted with the phosphatidylethanolamine in an organic solvent in the presence of an alkali catalyst.
  • the organic solvent includes halogenated hydrocarbon solvents such as chloroform, dichloromethane and dichloroethane; hydrocarbon solvents such as hexane and heptane; aromatic solvents such as benzene, toluene and xylene; ether solvents such as dimethyl ether, diethyl ether, methyl ethyl ether and tetrahydrofuran; and ester solvents such as ethyl acetate, butyl acetate and isobutyl acetate.
  • the alkali catalyst includes metal hydroxides such as sodium hydroxide and potassium hydroxide, as well as a pyridine compound, pyrrolidine compound, piperidine compound, morpholine compound, alkylamine compound, dialkylamine compound, trialkylamine compound, alcohol amine compound, dialcohol amine compound, trialcohol amine compound, N-alkyl alcohol amine compound, N,N'-dialkyl alcohol amine compound, cycloalkylamine compound, benzylamine compound and aniline compound.
  • the phosphatidylethanolamine (5) is dissolved in such organic solvent to a final concentration of about 1 to 20% w/v, preferably about 5 to 15% w/v.
  • the alkali catalyst is added thereto in an amount of 0.1 to 10 equivalents, preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine.
  • the carboxylic acid compound (6) anhydride is added thereto in an amount of 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the phosphatidylethanolamine.
  • the mixture is reacted at a temperature of 0 to 40°C, preferably 25 to 35°C and for 0.5 to 5 hours, preferably 1 to 3 hours, in the presence of an inert gas such as a nitrogen gas.
  • the carboxylic acid compound (6) is a free carboxylic acid
  • the phosphatidylethanolamine and the free carboxylic acid compound (6) are reacted for example with a condensing agent in the presence of an organic solvent.
  • an organic solvent the same solvent as described above is used, and as the condensing agent, carbodiimides such as N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride can be used.
  • the phosphatidylethanolamine (5) is dissolved in such organic solvents to a final concentration of about 1 to 20% w/v, preferably about 5 to 15% w/v. Then, the condensing agent is added thereto in an amount of 0.1 to 10 equivalents, preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine. Then, the free carboxylic acid compound (6) is added thereto in an amount of 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the phosphatidylethanolamine.
  • the mixture is reacted in the presence of an inert gas such as nitrogen gas or the like at a reaction temperature of 0 to 40°C, preferably 25 to 35°C and for 0.5 to 5 hours, preferably 1 to 3 hours.
  • a condensing auxiliary agent such as N-hydroxysuccinimide, N-hydroxysulfosuccinimide, 1-hydroxybenzotriazole or the like is preferably added because side reactions can be suppressed.
  • Such condensing auxiliary agent can be added together with the condensing agent described above, and the amount of the condensing auxiliary agent added is preferably 0.1 to 10 equivalents, more preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine.
  • R 1 , R 2 , R 3 , X, Y and m have the same meanings as defined above.
  • the present compound (1) can be obtained by reacting the phosphatidylethanolamine (5), the dicarboxylic acid derivative (7) or its anhydride and the nucleophilic species (4) with one another.
  • the compound of the formula (7) is a carboxylic acid anhydride
  • the phosphatidylethanolamine and an anhydride of the carboxylic acid represented by the formula (7) are reacted with an alkali catalyst in the presence of an organic solvent.
  • the reaction product can be reacted with the nucleophilic species (4) to afford the objective compound (1).
  • the nucleophilic species (4), the organic solvent and the alkali solvent can be the same as described above in the reactions (a) and (b), and the reaction can be carried out under the same conditions as described above in the reactions (a) and (b).
  • the same reaction as above also applies where the compound of the formula (6) is a free carboxylic acid. That is, the phosphatidylethanolamine is reacted with the free carboxylic acid represented by the formula (6), in the presence of a condensing agent in an organic solvent. Then, the reaction product can be reacted with the nucleophilic species (4) to afford the objective compound (1).
  • the nucleophilic species (4), the organic solvent and the condensing agent can be the same as described above in the reactions (a) and (b), and the reaction can be carried out under the same conditions as described above in the reactions (a) and (b).
  • phosphatidylethanolamine derivative represented by the formula (1) can be isolated and purified by ordinary methods, for example means such as filtration, extraction, washing, drying, concentration, recrystallization and various kinds of chromatography.
  • the phosphatidylethanolamine derivative (1) of the present invention is a novel compound and can, by taking advantage of the characteristics of the phospholipid, be applied to various pharmaceutical preparations or used as starting materials thereof. Specifically, the phosphatidylethanolamine derivative (1) can be used as a constituent of microparticles such as liposomes.
  • the present invention is described in more detail by reference to specific examples.
  • the phosphatidylethanolamine derivative (1) can be formed into a lipid composition.
  • other phospholipid include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine and phosphatidylglycerol.
  • the derivative (1) is more preferably phosphatidylcholine.
  • the phosphatidylcholine may be either naturally occurring ones or synthetic ones.
  • the phosphatidylcholine is particularly preferably dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, or palmitoyloleoylphosphatidylcholine.
  • compositional ratio denoted by the phosphatidylethanolamine derivative (1) : other phospholipids in the lipid composition is preferably 1 to 20 : 80 to 99 (molar ratio), more preferably 2 to 10 : 90 to 98 (molar ratio).
  • the lipid composition is preferably a composition containing other neutral lipid.
  • the neutral lipid includes cholesterol or derivatives thereof (cholesterol pullulan, DC -cholesterol and the like), among which cholesterol is preferable.
  • the lipid composition containing other neutral lipid is preferably a composition wherein ratio denoted by the phosphatidylethanolamine derivative (1) : other phospholipid : other neutral lipid ratio is 1 to 20 : 30 to 70 : 10 to 70 (molar ratio), more preferably 2 to 10 : 50 to 70 : 20 to 50 (molar ratio).
  • the lipid composition can contain a phosphatidylethanolamine dicarboxylic acid derivative represented by formula (2):
  • R 4 and R 5 independently represent an acyl group, and n is an integer of 1 to 10.
  • the acyl group represented by R 4 and R 5 includes the same acyl groups derived from saturated or unsaturated fatty acids as defined above in R 2 and R 3 , and is preferably a C12 to C22 acyl group.
  • the acyl group is more preferably a C14 to C20 acyl group, particularly preferably an acyl group derived from oleic acid, palmitic acid, stearic acid or myristic acid.
  • n is an arbitrary integer of 1 to 10, preferably 2 to 4.
  • the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) can also be synthesized by a method described in USP 4,534,899 or a modification thereto.
  • the lipid composition contains the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2)
  • the lipid composition is preferably a composition wherein the ratio denoted by derivative of the formula (1) : phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : other phospholipid : other neutral lipid is 0.8 to 16 : 0.2 to 4 : 30 to 70 : 10 to 70 (molar ratio), more preferably 1.6 to 8 : 0.4 to 2 : 50 to 70 : 20 to 50 (molar ratio).
  • the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) can be endowed with a function in molecular targeting by binding an in vivo target factor to a part thereof.
  • the in vivo target factor is not particularly limited insofar as it is a substance directed to a cell in the living body and capable of binding to a receptor or surface antigen present on the surface of a target cell, and specific examples include transferrin, folic acid, hyaluronic acid, a sugar chain, an antibody (preferably a monoclonal antibody) and antibody fragments (Fab, Fab', F(ab') 2 , Fc and the like), among which transferrin, particularly iron-bound holo-type transferrin, is preferable.
  • transferrin is used, the transferrin used may be a commercially available purified protein or a product produced by genetic recombination.
  • a terminal carboxyl group of the derivative represented by the formula (2) is preferably used.
  • the derivative represented by the formula (2) is reacted with carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N'-dicyclohexyl carbodiimide (DCC) or N,N'-diisopropyl carbodiimide, through which succinimide such as N-hydroxysulfosuccinimide (S-NHS) is bound to the derivative represented by the formula (2).
  • EDC N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • DCC N,N'-dicyclohexyl carbodiimide
  • S-NHS succinimide
  • the lipid composition contains a phospholipid having an in vivo target factor bound to the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2)
  • the lipid composition is preferably a composition wherein the ratio denoted by derivative of the formula (1) : phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : phospholipid having an in vivo target factor bound to the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : other phospholipid : other neutral lipid is 0.8 to 16 : 0.1998 to 3.96 : 0.002 to 0.04 : 30 to 70 : 10 to 70 (molar ratio), more preferably 1.6 to 8 : 0.39 to 1.98 : 50 to 70 : 20 to 50 (molar ratio).
  • Another mode of the invention provides a liposome having, as a lipid component, a phosphatidylethanolamine derivative represented by the formula (1).
  • the liposome is a spherical lipid bilayer having an aqueous portion inside.
  • the liposome upon formation, incorporates molecules in an aqueous solution into the aqueous portion inside.
  • the molecules incorporated into the liposome are protected from an external microenvironment, and the liposome is fused with a cell membrane, thus being transported efficiently into the cytoplasm.
  • Such liposome is not particularly limited insofar the phosphatidylethanolamine derivative represented by the formula (1) is contained as the lipid component, but preferably other phospholipid is also contained therein. Such other phospholipid and its compositional ratio in the liposome are the same as described in the above lipid composition.
  • the liposome of the present invention preferably contains other neutral lipid in addition to the other phospholipid.
  • Such other neutral lipid and its compositional ratio in the liposome are the same as described in the above lipid composition.
  • the liposome according to the present invention can contain the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) in addition to the other phospholipid and other neutral lipid.
  • Such phosphatidylethanolamine dicarboxylic acid derivative and its compositional ratio in the liposome are the same as described in the above lipid composition.
  • Such phosphatidylethanolamine dicarboxylic acid derivative can allow an in vivo target factor bound to at least a part thereof.
  • Such in vivo target factor and its compositional ratio in the liposome are the same as described in the above lipid composition.
  • the liposome of the present invention can have a drug, a nucleic acid or an inert gas incorporated therein to serve as a medical drug or a diagnostic drug.
  • the drug includes medical drugs or diagnostic drugs.
  • the medical drugs are not particularly limited insofar as they are those used for therapy and/ or prophylaxis of various diseases in mammals, preferably humans.
  • the medical drugs are more preferably antitumor substances, that is, anticancer agents.
  • anticancer agents include alkylating agents (nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan or the like; ethylene imine derivatives such as triethylenethiophosphoramide; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine, lomustine, semustine, streptozotocin; triazines such as dacarbazine; and metal salts such as carboplatin, cicplatin, oxaliplatin), antimetabolites (folate analogues such as methotrexate, raltitrexed, trimetrexate; pyrimidine analogues such as azacitidine, capecitabine, cytarabine, floxuridine, fluorouracil, gemcitabine; and purine analogues such as mercaptopurine, thioguanine
  • the diagnostic drugs are not particularly limited insofar as they can be used for diagnosis of various diseases in mammals, preferably humans.
  • Specific examples include X-ray contrast agents (nonionic monomer type contrast agents such as iopromide, iomeprol, iopamidol, ioversol, iohexol, ioxilan; nonionic dimer type contrast agents such as iotrolan, iodixanol; ionic monomer type contrast agents such as meglumine sodium amidotrizoate, sodium iotalamate, meglumine iotalamate; ionic dimer type contrast agents such as ioxaglic acid, iotroxate meglumine; oil agents such as iodized oil; and oral agents such as barium sulfate), MRI contrast agents (positive contrast agents such as gadodiamide hydrate, gadoteridol, meglumine gadoterate, meglumine gadopentate; and negative contrast
  • the nucleic acid includes a biological polymer consisting of nucleosides or nucleotides linked via ester linkages, and specific examples include cDNA, mRNA, antisense oligonucleotide, ribozyme, siRNA and the like. These nucleic acids are incorporated in an amount of 1 to 400 ⁇ g per mg of the total lipid mass constituting the liposome.
  • the inert gas includes, for example, inert gases known in the field of supersonic imaging agents, specifically perfluorocarbon gases such as perfluoromethane, perfluoroethane, perfluorobutane and perfluoropentane; hexafluoride sulfide gas; perfluoroether gas; nitrogen gas; and noble gases such as helium, argon and neon.
  • perfluorocarbon gases such as perfluoromethane, perfluoroethane, perfluorobutane and perfluoropentane
  • hexafluoride sulfide gas such as perfluoroethane, perfluorobutane and perfluoropentane
  • perfluorocarbon gases such as perfluoromethane, perfluoroethane, perfluorobutane and perfluoropentane
  • hexafluoride sulfide gas such as perfluoroethane, perfluorobut
  • the drug or nucleic acid described above can be used if necessary after dissolution in a solution of sugars such as trehalose, maltose, sucrose, lactose, mannitol, glycerol and dextrose.
  • concentration of such sugar solution is preferably about 10 wt% or less.
  • the liposome of the present invention can be produced by dissolving the phospholipid in a suitable organic solvent and then dispersing the resulting solution in an aqueous solution, followed by carrying out supersonic treatment or a reverse phase evaporation method.
  • the phospholipid is dissolved in a suitable organic solvent and then dispersed in an aqueous solution containing the drug or nucleic acid, followed by carrying out supersonic treatment or a reverse phase evaporation method.
  • the liposome For preparing the liposome, it is possible to employ not only a reverse phase evaporation method (REV method) (USP4,235,871) but also general methods of forming liposomes, such as a simple hydration method, an ethanol injection method or the like. Then, the liposomes thus formed can be fractionated according to their size to prepare liposomes having a uniform average particle diameter. If necessary, the liposome solution may be concentrated, and for this concentration, methods such as ultrafiltration, ultracentrifugation or the like can be utilized.
  • REV method reverse phase evaporation method
  • the in vivo target factor described above can be bound to the outer surface of the liposome.
  • a proteinous factor such as transferrin is used as the in vivo target factor
  • a terminal carboxyl group of the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) is preferably used.
  • the terminal carboxyl group is reacted with carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N'-dicyclohexyl carbodiimide (DCC) or N,N'-diisopropyl carbodiimide and then bound via this carbodiimide to succinimide such as N-hydroxysulfosuccinimide (S-NHS).
  • the liposome having this linker added thereto can be reacted with a proteinous factor such as transferrin, to give an in vivo factor-bound liposome that is a liposome having the proteinous factor bound to the outer surface thereof.
  • a proteinous factor such as transferrin
  • S-NHS is previously bound by the same method to a terminal carboxyl group of the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) and then converted, with other phospholipid or the like as the phospholipid, into a liposome to which an in vivo target factor can then be bound.
  • the liposome described above is previously prepared and placed in a closed container having a predetermined airspace.
  • the airspace is filled with the above-mentioned gas, and then the liposome is subjected to supersonic treatment, specifically by irradiation with a supersonic wave at 20 to 50 kHz for 1 to 5 minutes thereby replacing the aqueous solution filling inside the liposome by the inert gas, whereby a liposome having the gas incorporated therein can be easily and stably prepared.
  • the thus obtained liposome having a drug incorporated therein according to the present invention can be used by forming it into a pharmaceutical preparation with a pharmaceutically acceptable carrier.
  • Such liposome preparation is used preferably as a pharmaceutical composition for treating tumor.
  • the objective tumor includes, but is not limited to, nonsolid tumors such as leukemia, multiple myeloma, lymphoma or the like, as well as solid tumors represented by cancers in the bile duct, bone, bladder, brain/ CNS, breast, colon/ rectum, uterine mucosa, stomach, head/ cervical region, liver, lung, nerve cell, esophagus, ovary, spleen, prostate, kidney, skin, testis, thyroid gland, uterus, vagina or the like.
  • the pharmaceutically acceptable carrier sterilized water, a buffer solution and a saline solution can be used.
  • the pharmaceutical preparation may further contain various salts, sugars, proteins, starch, gelatin, vegetable oils, polyethylene glycol or the like.
  • Such liposome preparation may be administered parenterally by bolus injection or continuous injection.
  • the amount of the pharmaceutical composition to be administered is usually in the range of 10 to 100 mg/m 2 /day, though varying depending on the administration route, the severity of symptom, the age and condition of the patient, the degree of side effects or the like.
  • the liposome into which a diagnostic drug or an inert gas was incorporated can, together with a pharmaceutically acceptable carrier, be used as a diagnostic composition for X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, nuclear medicine imaging or the like.
  • MRI magnetic resonance imaging
  • the pharmaceutically acceptable carrier used may be the same as described above.
  • Such diagnostic composition can be administered parenterally by bolus injection or continuous injection.
  • the amount of the diagnostic composition to be administered is usually in the range of 10 to 100 mg/m 2 /day, though varying depending on the administration route, the severity of symptom, the age and condition of the patient, the degree of side effects or the like.
  • the liposome into which a nucleic acid was incorporated can, together with a pharmaceutically acceptable carrier, be similarly used as a composition.
  • the pharmaceutically acceptable carrier used may be the same as described above.
  • Such composition can be administered parenterally by bolus injection or continuous injection.
  • the amount of the diagnostic composition to be administered can vary but is usually in the range of 10 to 100 mg/m 2 /day.
  • N-glutaryl-dioleoylphosphatidylethanolamine (NOF Corporation) and a stirrer were placed into a 300-ml eggplant- shaped flask, and 75 mL each of chloroform and methanol were added thereto. 15 mL of 1 N sulfuric acid was added, and the mixture was subjected to esterification reaction by heating under reflux at 60°C for 24 hours.
  • the resulting concentrate was transferred to a 200-mL separatory funnel, and after 70 mL distilled water and 30 mL aqueous saturated sodium bicarbonate solution were added thereto, the mixture was shaken and only the lower layer was isolated and placed into a 300-mL eggplant- shaped flask. 25 mL each of chloroform and methanol were added to the residual liquid (upper layer) in the funnel, and after 70 mL distilled water and 30 mL aqueous saturated sodium bicarbonate solution were added thereto, the mixture was shaken and only the lower layer was similarly isolated and introduced into the 300-mL eggplant-shaped flask.
  • a developing solvent having the chloroform : methanol ratio of 5 : 1 was passed therethrough and fractions were collected in a total volume of 1000 mL (20 mL/ per fraction) which were then subjected to TLC (with the developing layer having the chloroform : methanol : distilled water ratio of 70 : 30 : 2), and a spot of the objective ester compound was confirmed.
  • the concentrate was dissolved in PBS (25 mM phosphate buffer and 125 mM NaCl, pH 7.4) and the particle-size distribution in the concentrate was measured by QELS (nano-ZS manufactured by Malvern), indicating that the average particle diameter was 119 nm (PDI 0.053, Z-ave-46.8).
  • DSPC and methotrexate of the liposome preparation were quantitatively measured by high performance liquid chromatography using a detector (UV); L-2400/ L-2420 manufactured by Hitachi, Ltd. and a detector (PAD/ DAD); L-2450 manufactured by Hitachi, Ltd., respectively.
  • the result obtained indicated that the content of methotrexate and DSPC was 16 ⁇ g and 1 mg, respectively.

Abstract

The present invention provides a liposome preparation having high efficiency of delivery of a drug into a target cell, which exhibits such an appropriate retention in blood as to allow the pharmaceutical preparation to be incorporated into a target cell but not such a long time as to induce its side effects on normal cells, as well as a novel synthetic phospholipid useful in preparation of the liposome preparation. Disclosed is a phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof: wherein X represents -0-, -NH- or -S-, R1 represents an alkyl group, a cycloalkyl group or an aryl group, R2 and R3 independently represent an acyl group, and m represents an integer of 1 to 10.

Description

PHOSPHATIDYLETHANOLAMINE DERIVATIVE AND LIPOSOME CONTAINING THE SAME
Cross Reference to Related Applications
[0001] This application claims the benefit of U.S. Provisional Pat. App. No. 60/842,589, filed September 5, 2006, the contents of which is hereby incorporated by reference in its entirety.
Technical Field
[0002] The present invention relates to a novel phosphatidylethanolamine derivative and a liposome containing the same.
Background Art
[0003] For reducing the side effects brought by a pharmaceutical preparation on normal cells and for protecting an unstable pharmaceutical preparation in a step of from the delivery thereof to its target cell, attempts on administering the pharmaceutical preparation by incorporating it into liposomes are intensively examined.
[0004] However, initial liposomes make use of naturally occurring cell membrane phospholipids such as egg-yolk phospholipids and soybean phospholipids which upon intravenous administration, are easily incorporated into the reticuloendothelial system such as the liver, spleen and thus there is a problem of low retention in blood.
[0005] As a means to solve this problem, liposomes (PEG liposomes) wherein synthetic phospholipids whose lipid moiety contains saturated bonds only are used as a constituent of the liposome membrane, and further, the surface of which is modified with polyethylene glycol have been developed and they have been gaining a growing attention as epoch-making liposomes which are highly retainable in blood without being incorporated into the cellular endothelial system. However, such a strong effort as to retain the liposomes in blood has created a problem of side effects such as the hand foot syndrome, that is a side effect brought by Doxil (liposome preparation of doxorubicin) affecting the peripheral system (as described in a package leaflet of Doxil). [0006] At present, there is a demand for further improvement in respect of the incorporation of pharmaceutical preparations into liposomes, the stability of such liposome preparation itself, suitable retainability in blood, and efficient delivery into a target cell.
[0007] A synthetic phospholipid compound having a dicarboxylic acid bound to phosphatidylethanolamine, which is useful for delivery of a pharmaceutical preparation, has been reported (Patent Document 1), but the compound described therein substantially has a polyalkylene glycol moiety, and the problem of the above side effects is a concern.
[Patent Document 1] JP-A-59-204198
Disclosure of the Invention
[0008] The object of the present invention is to provide a liposome preparation having high efficiency of delivery of a drug into a target cell which exhibits such an appropriate retention in blood as to allow the pharmaceutical preparation to be incorporated into a target cell but not such a long time as to induce its side effects on normal cells, as well as a novel synthetic phospholipid useful in preparation of the liposome preparation.
[0009] Under these circumstances, the present inventors made extensive study for solving the problem described above, and as a result they found that a specific phosphatidylethanolamine derivative represented by the general formula (1) below can reduce the unbalance in the distribution of electric charges on the surfaces of liposomes and is thus useful as synthetic phospholipid for the liposome preparation described above, and the present invention was thereby completed.
[0010] That is, the present invention provides a phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof:
[Chemical Formula] H2C-OR2
RJO— CH O O O
CH2-O-P-O-CH2CH2-N-C-(CH2)^-C-XR1
OH
( 1 ) wherein X represents -O-, -NH- or -S-, R1 represents an alkyl group, a cycloalkyl group or an aryl group, R2 and R3 independently represent an acyl group, and m represents an integer of 1 to 10, as well as a liposome containing the same.
[0011] The present invention also provides a lipid composition containing the phosphatidylethanolamine derivative (1) described above and other phospholipid.
Effect of the Invention
[0012] Liposomes using the novel phospholipid of the present invention contain a pharmaceutical preparation such as an anticancer agent, have high storage stability, are delivered highly efficiently into a target cell upon administration, and are retained in blood for an appropriate time to allow the pharmaceutical preparation to be incorporated into a target cell but not retained in blood for such a long time as to induce their side effects on normal cells.
Brief Description of Drawing
[0013] Fig. 1 shows a 1H-NMR chart of the phospholipid obtained in Example 1.
Best Mode for Carrying Out the Invention
[0014] The alkyl group represented by R1 in the general formula (1) includes Cl to ClO alkyl groups, and specific examples thereof include a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, t-butyl group, n-pentyl group, n-hexyl group and the like. [0015] The cycloalkyl group includes C3 to ClO cycloalkyl groups, and specific examples thereof include a cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group and the like.
[0016] The aryl group includes C6 to Cl 5 aryl groups, and specific examples thereof include a phenyl group, tolyl group, xylyl group, 1-naphtyl group, 2-naphtyl group and the like.
[0017] Among these substituent groups, R1 is preferably an alkyl group or an aryl group, particularly preferably an alkyl group.
[0018] X is preferably -O- or -NH-, particularly preferably -O-.
[0019] The acyl group represented by R2 and R3 is preferably an acyl group derived from a saturated or unsaturated fatty acid, more preferably a C 12 to C22 acyl group derived from a fatty acid, more preferably a C14 to C20 acyl group derived from a fatty acid. Examples of the fatty acids from which these acyl groups are derived include linear saturated carboxylic acids such as lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, tuberculostearic acid, arachidic acid, heneicosanoic acid and behenic acid, as well as linear unsaturated carboxylic acids such as 2-lauroleic acid, linderic acid, Tohaku acid, 5-lauroleic acid, 11-lauroleic acid, tsuzuic acid, 5-miristoleic acid, myristoleic acid, 2-palmitoleic acid, 7-palmitoleic acid, cis-9-palmitoleic acid, trans-9-palmitoleic acid, petroselinic acid, petroselidinic acid, oleic acid, elaidic acid, asclepinic acid, vaccenic acid, gondoic acid, trans-gondoic acid, erucic acid, linolic acid, linoelaidic acid, α-eleostearic acid, β-eleostearic acid, linolenic acid, linolenelaidic acid, α-parinaric acid, β-parinaric acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, gadoleic acid, clupanodonic acid, stearidonic acid, punicic acid, ricinoleic acid, ricinelaidic acid and the like. Each of R2 and R3 is particularly preferably an acyl group derived from oleic acid, palmitic acid, stearic acid or myristic acid.
[0020] m is an arbitrary integer of 1 to 10, preferably 2 to 4. [0021] A salt of the phosphatidylethanolamine derivative (1) includes alkali metal salts such as sodium salt, potassium salt and the like, alkaline earth metal salts such as calcium salt, magnesium salt and the like, amine salts, ammonium salts and the like.
[0022] The phosphatidylethanolamine derivative (1) of the present invention can be produced for example by the following methods (a) to (c):
[Chemical Formula 2]
Reaction (a):
f&JS ( a ) :
H2C-OR2
R3O-CH O O O
CH2-O- P— O— CH2CH2-N- C— (CH2) - C-OH + R1— X— Y
OH
( 3 ) ( 4 )
H2C-OR2 R3O-CH O O O *~ CH2-O-P-O-CH2CH2- -NN--C-(CH2)^-C-XR1 H
OH
( D
wherein Y represents a hydrogen atom or a metal atom, and R1, R2, R3, X and m have the same meanings as defined above.
[0023] That is, a phosphatidylethanolamine dicarboxylic acid derivative represented by the general formula (3) is reacted with a nucleophilic species (4) to give the present compound (1).
[0024] The phosphatidylethanolamine dicarboxylic acid derivative (3) can be produced by a method described in USP 4,534,899 or a modification thereto. [0025] Y in the nucleophilic species (4) includes a hydrogen atom, a metal (MgBr and the like) constituting a Grignard reagent, a metal (CuLi and the like) constituting a Gillman reagent. The compound represented by R1XH includes saturated aliphatic hydrocarbon alcohols such as methanol, ethanol; saturated alicyclic hydrocarbon alcohols such as cyclohexanol; aromatic hydrocarbon alcohols such as phenol; saturated aliphatic hydrocarbon amines such as methylamine, ethylamine; saturated alicyclic hydrocarbon amines such as cyclohexylamine; aromatic hydrocarbon amines such as aniline; saturated aliphatic hydrocarbon thiols such as methyl mercaptan; saturated alicyclic hydrocarbon thiols such as cyclohexyl mercaptan; and aromatic hydrocarbon thiols such as thiophenol.
[0026] The phosphatidylethanolamine derivative (1) of the invention can be obtained by reacting 50 to 50,000 equivalents, preferably 1,000 to 5,000 equivalents, of the nucleophilic species (4) with the carboxylic acid compound of the formula (3) in the presence of an organic solvent such as chloroform, methylene chloride or the like and a catalyst such as triethyl amine, potassium carbonate, sulfuric acid or the like, at a temperature not higher than the boiling point of the nucleophilic species (4). The amount of the organic solvent used is preferably about half (volume ratio) of the nucleophilic species (4), and the amount of the catalyst added is preferably 1 to 10 equivalents relative to the carboxylic acid compound (3).
[0027] Depending on the nucleophilic species (4) used, the phosphatidylethanolamine derivative (1) can be produced by converting the carboxylic acid compound of the formula (3) into the corresponding halide by a halogenating agent such as thionyl chloride, phosphoryl chloride or the like and then reacting it with the nucleophilic species (4). Specifically, the carboxylic acid compound of the formula (3) is reacted with 2 to 5 equivalents of the halogenating agent in the presence of an organic solvent such as chloroform, methylene chloride or the like, to give a halide of the carboxylic acid compound. The amount of the organic solvent used is preferably 1,000 to 5,000 equivalents relative to the carboxylic acid compound of the formula (3). Subsequently, this halide is reacted with the nucleophilic species (4). Specifically, the halide is reacted with 1 to 5 equivalents, preferably 1.5 to 2 equivalents, of the nucleophilic species (4) at 0°C to room temperature in the presence of an organic solvent such as chloroform, methylene chloride or the like and a catalyst such as triethylamine, potassium carbonate or the like, whereby the phosphatidylethanolamine derivative (1) of the present invention can be obtained. The amount of the organic solvent used is preferably 1,000 to 5,000 equivalents relative to the carboxylic acid compound of the formula (3), and the amount of the catalyst added is preferably 1 to 5 equivalents relative to the nucleophilic species (4).
[Chemical Formula 3]
Reaction (b):
Rfc ( b ) :
H2C-OR2 R3O-CH O
CH2-O-P-O-CH2CH2NH2 + HOOC(CH2)^- COXR1
OH f a λ
( 5 ) ( 6 )
H2C-OR2 I
RJO— CH O O O
I
CH2-O-P-O-CH2CH2-N-C-(CH2)^-C-XR1 I H
OH
( D wherein R1, R2, R3, X and m have the same meanings as defined above.
[0028] That is, the present compound (1) can be obtained by reacting the carboxylic acid compound (6) or its anhydride with the phosphatidylethanolamine (5).
[0029] When the carboxylic acid compound (6) is a carboxylic acid anhydride, the carboxylic acid compound (6) anhydride is reacted with the phosphatidylethanolamine in an organic solvent in the presence of an alkali catalyst. The organic solvent includes halogenated hydrocarbon solvents such as chloroform, dichloromethane and dichloroethane; hydrocarbon solvents such as hexane and heptane; aromatic solvents such as benzene, toluene and xylene; ether solvents such as dimethyl ether, diethyl ether, methyl ethyl ether and tetrahydrofuran; and ester solvents such as ethyl acetate, butyl acetate and isobutyl acetate. Such organic solvents are more preferably those wherein active hydrogens are eliminated or reduced by sufficient dehydrogenation and the like. The alkali catalyst includes metal hydroxides such as sodium hydroxide and potassium hydroxide, as well as a pyridine compound, pyrrolidine compound, piperidine compound, morpholine compound, alkylamine compound, dialkylamine compound, trialkylamine compound, alcohol amine compound, dialcohol amine compound, trialcohol amine compound, N-alkyl alcohol amine compound, N,N'-dialkyl alcohol amine compound, cycloalkylamine compound, benzylamine compound and aniline compound.
[0030] For example, the phosphatidylethanolamine (5) is dissolved in such organic solvent to a final concentration of about 1 to 20% w/v, preferably about 5 to 15% w/v. Then, the alkali catalyst is added thereto in an amount of 0.1 to 10 equivalents, preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine. Then, the carboxylic acid compound (6) anhydride is added thereto in an amount of 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the phosphatidylethanolamine. The mixture is reacted at a temperature of 0 to 40°C, preferably 25 to 35°C and for 0.5 to 5 hours, preferably 1 to 3 hours, in the presence of an inert gas such as a nitrogen gas.
[0031] When the carboxylic acid compound (6) is a free carboxylic acid, the phosphatidylethanolamine and the free carboxylic acid compound (6) are reacted for example with a condensing agent in the presence of an organic solvent. As the organic solvent, the same solvent as described above is used, and as the condensing agent, carbodiimides such as N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride can be used.
[0032] Specifically, the phosphatidylethanolamine (5) is dissolved in such organic solvents to a final concentration of about 1 to 20% w/v, preferably about 5 to 15% w/v. Then, the condensing agent is added thereto in an amount of 0.1 to 10 equivalents, preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine. Then, the free carboxylic acid compound (6) is added thereto in an amount of 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the phosphatidylethanolamine. The mixture is reacted in the presence of an inert gas such as nitrogen gas or the like at a reaction temperature of 0 to 40°C, preferably 25 to 35°C and for 0.5 to 5 hours, preferably 1 to 3 hours. In this reaction, a condensing auxiliary agent such as N-hydroxysuccinimide, N-hydroxysulfosuccinimide, 1-hydroxybenzotriazole or the like is preferably added because side reactions can be suppressed. Such condensing auxiliary agent can be added together with the condensing agent described above, and the amount of the condensing auxiliary agent added is preferably 0.1 to 10 equivalents, more preferably 0.5 to 5 equivalents, relative to the phosphatidylethanolamine.
[Chemical Formula 4]
Reaction (c):
®S ( c ) :
H2C-OR2
R3O-CH O
I I I
CH2-O-P-O-CH2CH2NH2 + HOOC(CH2^nCOOH + R1XY
0H ( 5 ) ( 7 ) ( 4 )
H2C-OR2
R3O-CH O O O I I l I l I l *~ CH2- O— P— O— CH2CH2-N- C— (CH2) - C— XR1
I H
OH
( D
wherein R1, R2, R3, X, Y and m have the same meanings as defined above.
[0033] That is, the present compound (1) can be obtained by reacting the phosphatidylethanolamine (5), the dicarboxylic acid derivative (7) or its anhydride and the nucleophilic species (4) with one another.
[0034] When the compound of the formula (7) is a carboxylic acid anhydride, the phosphatidylethanolamine and an anhydride of the carboxylic acid represented by the formula (7) are reacted with an alkali catalyst in the presence of an organic solvent. Then, the reaction product can be reacted with the nucleophilic species (4) to afford the objective compound (1). The nucleophilic species (4), the organic solvent and the alkali solvent can be the same as described above in the reactions (a) and (b), and the reaction can be carried out under the same conditions as described above in the reactions (a) and (b).
[0035] The same reaction as above also applies where the compound of the formula (6) is a free carboxylic acid. That is, the phosphatidylethanolamine is reacted with the free carboxylic acid represented by the formula (6), in the presence of a condensing agent in an organic solvent. Then, the reaction product can be reacted with the nucleophilic species (4) to afford the objective compound (1). The nucleophilic species (4), the organic solvent and the condensing agent can be the same as described above in the reactions (a) and (b), and the reaction can be carried out under the same conditions as described above in the reactions (a) and (b).
[0036] The thus obtained phosphatidylethanolamine derivative represented by the formula (1) can be isolated and purified by ordinary methods, for example means such as filtration, extraction, washing, drying, concentration, recrystallization and various kinds of chromatography.
[0037] The phosphatidylethanolamine derivative (1) of the present invention is a novel compound and can, by taking advantage of the characteristics of the phospholipid, be applied to various pharmaceutical preparations or used as starting materials thereof. Specifically, the phosphatidylethanolamine derivative (1) can be used as a constituent of microparticles such as liposomes. Hereinafter, the present invention is described in more detail by reference to specific examples.
[0038] The phosphatidylethanolamine derivative (1), together with other phospholipid, can be formed into a lipid composition. Preferable examples of such other phospholipid include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine and phosphatidylglycerol. The derivative (1) is more preferably phosphatidylcholine. The phosphatidylcholine may be either naturally occurring ones or synthetic ones. The phosphatidylcholine is particularly preferably dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, or palmitoyloleoylphosphatidylcholine.
[0039] The compositional ratio denoted by the phosphatidylethanolamine derivative (1) : other phospholipids in the lipid composition is preferably 1 to 20 : 80 to 99 (molar ratio), more preferably 2 to 10 : 90 to 98 (molar ratio).
[0040] The lipid composition is preferably a composition containing other neutral lipid. The neutral lipid includes cholesterol or derivatives thereof (cholesterol pullulan, DC -cholesterol and the like), among which cholesterol is preferable. The lipid composition containing other neutral lipid is preferably a composition wherein ratio denoted by the phosphatidylethanolamine derivative (1) : other phospholipid : other neutral lipid ratio is 1 to 20 : 30 to 70 : 10 to 70 (molar ratio), more preferably 2 to 10 : 50 to 70 : 20 to 50 (molar ratio).
[0041] The lipid composition can contain a phosphatidylethanolamine dicarboxylic acid derivative represented by formula (2):
[Chemical Formula 5]
H2C-OR4
R5O-CH O O O
I I l I l I l
CH2 z-O-P . -O-CH2 zCH2 z-N-C-( vCH2 Z)J j —i C— OH
OH
( 2 )
wherein R4 and R5 independently represent an acyl group, and n is an integer of 1 to 10.
[0042] The acyl group represented by R4 and R5 includes the same acyl groups derived from saturated or unsaturated fatty acids as defined above in R2 and R3, and is preferably a C12 to C22 acyl group. The acyl group is more preferably a C14 to C20 acyl group, particularly preferably an acyl group derived from oleic acid, palmitic acid, stearic acid or myristic acid. [0043] n is an arbitrary integer of 1 to 10, preferably 2 to 4.
[0044] The phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) can also be synthesized by a method described in USP 4,534,899 or a modification thereto.
[0045] When the lipid composition contains the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2), the lipid composition is preferably a composition wherein the ratio denoted by derivative of the formula (1) : phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : other phospholipid : other neutral lipid is 0.8 to 16 : 0.2 to 4 : 30 to 70 : 10 to 70 (molar ratio), more preferably 1.6 to 8 : 0.4 to 2 : 50 to 70 : 20 to 50 (molar ratio).
[0046] The phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) can be endowed with a function in molecular targeting by binding an in vivo target factor to a part thereof.
[0047] The in vivo target factor is not particularly limited insofar as it is a substance directed to a cell in the living body and capable of binding to a receptor or surface antigen present on the surface of a target cell, and specific examples include transferrin, folic acid, hyaluronic acid, a sugar chain, an antibody (preferably a monoclonal antibody) and antibody fragments (Fab, Fab', F(ab')2, Fc and the like), among which transferrin, particularly iron-bound holo-type transferrin, is preferable. When transferrin is used, the transferrin used may be a commercially available purified protein or a product produced by genetic recombination.
[0048] As a means of binding an in vivo target factor to the phosphatidylethanolamine represented by the formula (2), a terminal carboxyl group of the derivative represented by the formula (2) is preferably used. Specifically, the derivative represented by the formula (2) is reacted with carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N'-dicyclohexyl carbodiimide (DCC) or N,N'-diisopropyl carbodiimide, through which succinimide such as N-hydroxysulfosuccinimide (S-NHS) is bound to the derivative represented by the formula (2). Then, this product is reacted with an in vivo target factor, whereby the lipid derivative having the in vivo target factor bound to the terminal carboxyl group thereof can be obtained.
[0049] When the lipid composition contains a phospholipid having an in vivo target factor bound to the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2), the lipid composition is preferably a composition wherein the ratio denoted by derivative of the formula (1) : phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : phospholipid having an in vivo target factor bound to the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) : other phospholipid : other neutral lipid is 0.8 to 16 : 0.1998 to 3.96 : 0.002 to 0.04 : 30 to 70 : 10 to 70 (molar ratio), more preferably 1.6 to 8 : 0.39 to 1.98 : 50 to 70 : 20 to 50 (molar ratio).
[0050] Another mode of the invention provides a liposome having, as a lipid component, a phosphatidylethanolamine derivative represented by the formula (1).
[0051] The liposome is a spherical lipid bilayer having an aqueous portion inside. The liposome, upon formation, incorporates molecules in an aqueous solution into the aqueous portion inside. The molecules incorporated into the liposome are protected from an external microenvironment, and the liposome is fused with a cell membrane, thus being transported efficiently into the cytoplasm.
[0052] Such liposome is not particularly limited insofar the phosphatidylethanolamine derivative represented by the formula (1) is contained as the lipid component, but preferably other phospholipid is also contained therein. Such other phospholipid and its compositional ratio in the liposome are the same as described in the above lipid composition.
[0053] The liposome of the present invention preferably contains other neutral lipid in addition to the other phospholipid. Such other neutral lipid and its compositional ratio in the liposome are the same as described in the above lipid composition.
[0054] The liposome according to the present invention can contain the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) in addition to the other phospholipid and other neutral lipid. Such phosphatidylethanolamine dicarboxylic acid derivative and its compositional ratio in the liposome are the same as described in the above lipid composition. Such phosphatidylethanolamine dicarboxylic acid derivative can allow an in vivo target factor bound to at least a part thereof. Such in vivo target factor and its compositional ratio in the liposome are the same as described in the above lipid composition.
[0055] The liposome of the present invention can have a drug, a nucleic acid or an inert gas incorporated therein to serve as a medical drug or a diagnostic drug.
[0056] The drug includes medical drugs or diagnostic drugs. The medical drugs are not particularly limited insofar as they are those used for therapy and/ or prophylaxis of various diseases in mammals, preferably humans. The medical drugs are more preferably antitumor substances, that is, anticancer agents. Specifically, such anticancer agents include alkylating agents (nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan or the like; ethylene imine derivatives such as triethylenethiophosphoramide; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine, lomustine, semustine, streptozotocin; triazines such as dacarbazine; and metal salts such as carboplatin, cicplatin, oxaliplatin), antimetabolites (folate analogues such as methotrexate, raltitrexed, trimetrexate; pyrimidine analogues such as azacitidine, capecitabine, cytarabine, floxuridine, fluorouracil, gemcitabine; and purine analogues such as mercaptopurine, thioguanine, pentostatin, cladribine, fludarabine), natural product-derived anticancer agents (cell division inhibitors such as vinblastine, vincristine, vindesine, vinorelbine; microtubule polymer stabilizers such as docetaxel, paclitaxel; topoisomerase I inhibitors such as irinotecan, topotecan; topoisomerase II inhibitors such as etoposide, teniposide; antibiotics such as bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, plicamycin, mitomycin, mitoxantrone; and enzyme preparations such as asparaginase), hormones and hormone antagonists (androgens such as fluoxymesterone; corticosteroids such as dexamethasone, prednisone; estrogens such as diethylstilbestrol; progestins such as megestrol acetate, medroxyprogesterone acetate; estrogen antagonists such as raloxifene, tamoxifen, toremifene; aromatase inhibitors such as aminoglutethimide, anastrozole, letrozole; androgen antagonists such as bicalutamide, flutamide, nilutamide; hypothalamic hormone analogues such as leuprolide, goserelin; and thyroid hormones such as levothyroxine, liothyronine), biological preparations (monoclonal antibodies such as trastuzumab, rituximab; interferons such as interferon-α2a, interferon- oc2t,; interleukins such as aldesleukin, oprelvekin; and myeloid and erythroid stimulating factors such as erythropoietin, filgrastim, sargramostim), urea derivatives such as hydroxyurea, methyl hydrazine derivatives such as procarbazine, adrenocortical inhibitors such as mitotane, substituted melamine derivatives such as altretamine, acridine dyes such as amsacrine, bisphosphonates such as pamidronate, photosensitive agents such as porfimer, site protectors such as amifostine, platelet-decreasing agents such as anagrelide, and somatostatin analogues such as octreotide.
[0057] The diagnostic drugs are not particularly limited insofar as they can be used for diagnosis of various diseases in mammals, preferably humans. Specific examples include X-ray contrast agents (nonionic monomer type contrast agents such as iopromide, iomeprol, iopamidol, ioversol, iohexol, ioxilan; nonionic dimer type contrast agents such as iotrolan, iodixanol; ionic monomer type contrast agents such as meglumine sodium amidotrizoate, sodium iotalamate, meglumine iotalamate; ionic dimer type contrast agents such as ioxaglic acid, iotroxate meglumine; oil agents such as iodized oil; and oral agents such as barium sulfate), MRI contrast agents (positive contrast agents such as gadodiamide hydrate, gadoteridol, meglumine gadoterate, meglumine gadopentate; and negative contrast agents such as ferric ammonium citrate, ferucarbotran, ferumoxides), magnetic nanoparticles such as magnetite, and in vivo diagnostic radioactive drugs for PET such as 18F-fluorodeoxyglucose. These drugs are incorporated in an amount of 3 to 400 μg per mg of the total lipid mass constituting the liposome.
[0058] The nucleic acid includes a biological polymer consisting of nucleosides or nucleotides linked via ester linkages, and specific examples include cDNA, mRNA, antisense oligonucleotide, ribozyme, siRNA and the like. These nucleic acids are incorporated in an amount of 1 to 400 μg per mg of the total lipid mass constituting the liposome.
[0059] The inert gas includes, for example, inert gases known in the field of supersonic imaging agents, specifically perfluorocarbon gases such as perfluoromethane, perfluoroethane, perfluorobutane and perfluoropentane; hexafluoride sulfide gas; perfluoroether gas; nitrogen gas; and noble gases such as helium, argon and neon.
[0060] The drug or nucleic acid described above can be used if necessary after dissolution in a solution of sugars such as trehalose, maltose, sucrose, lactose, mannitol, glycerol and dextrose. The concentration of such sugar solution is preferably about 10 wt% or less.
[0061] The liposome of the present invention can be produced by dissolving the phospholipid in a suitable organic solvent and then dispersing the resulting solution in an aqueous solution, followed by carrying out supersonic treatment or a reverse phase evaporation method. When the drug or nucleic acid is to be contained in the liposome, the phospholipid is dissolved in a suitable organic solvent and then dispersed in an aqueous solution containing the drug or nucleic acid, followed by carrying out supersonic treatment or a reverse phase evaporation method. For preparing the liposome, it is possible to employ not only a reverse phase evaporation method (REV method) (USP4,235,871) but also general methods of forming liposomes, such as a simple hydration method, an ethanol injection method or the like. Then, the liposomes thus formed can be fractionated according to their size to prepare liposomes having a uniform average particle diameter. If necessary, the liposome solution may be concentrated, and for this concentration, methods such as ultrafiltration, ultracentrifugation or the like can be utilized.
[0062] The in vivo target factor described above can be bound to the outer surface of the liposome. For example, when a proteinous factor such as transferrin is used as the in vivo target factor, a terminal carboxyl group of the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) is preferably used. That is, the terminal carboxyl group is reacted with carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N'-dicyclohexyl carbodiimide (DCC) or N,N'-diisopropyl carbodiimide and then bound via this carbodiimide to succinimide such as N-hydroxysulfosuccinimide (S-NHS). Then, the liposome having this linker added thereto can be reacted with a proteinous factor such as transferrin, to give an in vivo factor-bound liposome that is a liposome having the proteinous factor bound to the outer surface thereof.
[0063] Alternatively, S-NHS is previously bound by the same method to a terminal carboxyl group of the phosphatidylethanolamine dicarboxylic acid derivative represented by the formula (2) and then converted, with other phospholipid or the like as the phospholipid, into a liposome to which an in vivo target factor can then be bound.
[0064] When an inert gas is to be incorporated into the liposome, the liposome described above is previously prepared and placed in a closed container having a predetermined airspace. The airspace is filled with the above-mentioned gas, and then the liposome is subjected to supersonic treatment, specifically by irradiation with a supersonic wave at 20 to 50 kHz for 1 to 5 minutes thereby replacing the aqueous solution filling inside the liposome by the inert gas, whereby a liposome having the gas incorporated therein can be easily and stably prepared.
[0065] The thus obtained liposome having a drug incorporated therein according to the present invention can be used by forming it into a pharmaceutical preparation with a pharmaceutically acceptable carrier. Such liposome preparation is used preferably as a pharmaceutical composition for treating tumor. Specifically, the objective tumor includes, but is not limited to, nonsolid tumors such as leukemia, multiple myeloma, lymphoma or the like, as well as solid tumors represented by cancers in the bile duct, bone, bladder, brain/ CNS, breast, colon/ rectum, uterine mucosa, stomach, head/ cervical region, liver, lung, nerve cell, esophagus, ovary, spleen, prostate, kidney, skin, testis, thyroid gland, uterus, vagina or the like.
[0066] As the pharmaceutically acceptable carrier, sterilized water, a buffer solution and a saline solution can be used. The pharmaceutical preparation may further contain various salts, sugars, proteins, starch, gelatin, vegetable oils, polyethylene glycol or the like. Such liposome preparation may be administered parenterally by bolus injection or continuous injection. The amount of the pharmaceutical composition to be administered is usually in the range of 10 to 100 mg/m2/day, though varying depending on the administration route, the severity of symptom, the age and condition of the patient, the degree of side effects or the like.
[0067] The liposome into which a diagnostic drug or an inert gas was incorporated can, together with a pharmaceutically acceptable carrier, be used as a diagnostic composition for X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging, nuclear medicine imaging or the like. The pharmaceutically acceptable carrier used may be the same as described above. Such diagnostic composition can be administered parenterally by bolus injection or continuous injection. The amount of the diagnostic composition to be administered is usually in the range of 10 to 100 mg/m2/day, though varying depending on the administration route, the severity of symptom, the age and condition of the patient, the degree of side effects or the like.
[0068] The liposome into which a nucleic acid was incorporated can, together with a pharmaceutically acceptable carrier, be similarly used as a composition. The pharmaceutically acceptable carrier used may be the same as described above. Such composition can be administered parenterally by bolus injection or continuous injection. The amount of the diagnostic composition to be administered can vary but is usually in the range of 10 to 100 mg/m2/day.
EXAMPLES
[0069] Hereinafter, the present invention is described specifically by reference to the Examples, but these examples are not intended to limit the scope of the invention.
Example 1
Synthesis of methyl ester of N-glutarylphosphatidylethanolamine
[0070] 2.5 g of N-glutaryl-dioleoylphosphatidylethanolamine (NOF Corporation) and a stirrer were placed into a 300-ml eggplant- shaped flask, and 75 mL each of chloroform and methanol were added thereto. 15 mL of 1 N sulfuric acid was added, and the mixture was subjected to esterification reaction by heating under reflux at 60°C for 24 hours. After completion of the reaction was confirmed by TLC (with chloroform : methanol : distilled water = 70 : 30 : 2 on the developing layer), the flask was cooled to room temperature, and after the stirrer was removed, the reaction mixture was concentrated to a volume of about 100 mL by a rotary evaporator (N-IOOOV manufactured by EYELA).
[0071] The resulting concentrate was transferred to a 200-mL separatory funnel, and after 70 mL distilled water and 30 mL aqueous saturated sodium bicarbonate solution were added thereto, the mixture was shaken and only the lower layer was isolated and placed into a 300-mL eggplant- shaped flask. 25 mL each of chloroform and methanol were added to the residual liquid (upper layer) in the funnel, and after 70 mL distilled water and 30 mL aqueous saturated sodium bicarbonate solution were added thereto, the mixture was shaken and only the lower layer was similarly isolated and introduced into the 300-mL eggplant-shaped flask. This operation was repeated 3 times in total, and the absence of the objective ester compound in the aqueous layer was confirmed by TLC (with chloroform : methanol : distilled water = 70 : 30 : 2 on the developing layer), and pH of the extracted organic layer was measured (determined pH: about 3) and then the liquid was concentrated and dried by a rotary evaporator (N-IOOOV manufactured by EYELA).
[0072] An open column of 3 cm in diameter and 30 cm in height was charged with silica gel (Kieselgel 6OF254, layer thickness of 0.2 mm, manufactured by Merck) in chloroform until the gel reached 15 cm in height in the column. The concentrated and dried compound was dissolved in 3 to 5 mL chloroform and then applied onto the silica gel column. 100 mL chloroform was passed therethrough and collected as one fraction, and then a developing solvent having the chloroform : methanol ratio of 10 : 1 was passed therethrough to collect fractions in a total volume of 500 mL (20 mL/ per fraction) which were then subjected to TLC (with the ratio of chloroform : methanol : distilled water = 70 : 30 : 2) to confirm a spot of the objective ester compound. The presence objective ester compound was confirmed in the third fraction (i.e., at the time a total of 300 mL chloroform had been flown through), so fractions thereafter were collected and concentrated respectively.
[0073] A developing solvent having the chloroform : methanol ratio of 5 : 1 was passed therethrough and fractions were collected in a total volume of 1000 mL (20 mL/ per fraction) which were then subjected to TLC (with the developing layer having the chloroform : methanol : distilled water ratio of 70 : 30 : 2), and a spot of the objective ester compound was confirmed.
[0074] The collected and concentrated sample was dissolved in chloroform and transferred to a previously weighed eggplant-shaped flask and concentrated in rotary evaporator (N-IOOOV manufactured by EYELA). The sample was vacuum-dried overnight to give 250 mg objective ester compound.
[0075] The 1H-NMR measurement result measured in a heavy chloroform solution is shown in Fig. 1.
Example 2
Preparation example of liposome preparation
[0076] 91 mg distearoylphosphatidylcholine (DSPC manufactured by NOF Corporation), 25 mg cholesterol (manufactured by Solvey) and 9 mg of the ester compound synthesized by Example 1 (these ingredients were in the molar ratio of 62 : 33 : 5) were introduced into a brown vial with a volume of 13.5 mL. 500 μL ethanol was added thereto, then the mixture was stirred at 60°C for 15 minutes, 4.5 mL of 300 mM sodium bicarbonate, pH 8.9 was added thereto, and the mixture was stirred similarly at 60°C for 15 minutes.
[0077] Three sheets of 100-nm membrane filter (Track-Etch Membrane manufactured by Whatman) were stacked on one another, and set in an extruder (T-002 manufactured by NLI) for sizing the above mixture at 60°C. The sized liposome solution was subjected to ultrafiltration together with 145 mM sodium chloride solution (washing with the solution in 10-fold volume excess (50 mL)) and then concentrated to a volume of 7 mL.
[0078] The concentrate was dissolved in PBS (25 mM phosphate buffer and 125 mM NaCl, pH 7.4) and the particle-size distribution in the concentrate was measured by QELS (nano-ZS manufactured by Malvern), indicating that the average particle diameter was 119 nm (PDI 0.053, Z-ave-46.8).
[0079] 3 mL of 220 mM methotrexate solution (manufactured by Heumann PCS; a solution prepared by dissolving with NaOH and HCl and subsequently adjusted to pH 6.9) was added to the concentrate and stirred at 60°C for 2 days. The sample thus obtained was subjected to ultrafiltration together with 145 mM sodium chloride solution (washing with the solution in 10-fold volume excess (100 mL)) to give a liposome preparation having methothrexate incorporated therein.
[0080] DSPC and methotrexate of the liposome preparation were quantitatively measured by high performance liquid chromatography using a detector (UV); L-2400/ L-2420 manufactured by Hitachi, Ltd. and a detector (PAD/ DAD); L-2450 manufactured by Hitachi, Ltd., respectively. The result obtained indicated that the content of methotrexate and DSPC was 16 μg and 1 mg, respectively.

Claims

1. A phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof:
[Chemical Formula 1]
H2C-OR2
R3O-CH O O O
I I l I l I l
CH2-O-P-O-CH2CH2-N-C-(CH2)^-C-XR1 I H
OH
( D
wherein X represents -0-, -NH- or -S-, R1 represents an alkyl group, a cycloalkyl group or an aryl group, R2 and R3 independently represent an acyl group, and m represents an integer of 1 to 10.
2. The compound according to claim 1, wherein R2 and R3 independently represent a C 12 to C22 acyl group derived from a saturated or unsaturated fatty acid.
3. The compound according to claim 1 or 2, wherein m is an integer of 2 to 4.
4. The compound according to any one of claims 1 to 3, wherein X is -O- or -NH-, and R1 is an alkyl group or an aryl group.
5. The compound according to any one of claims 1 to 4, wherein X is -O-, and R1 is an alkyl group.
6. A lipid composition comprising a phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof and other phospholipid:
[Chemical Formula 2] H2C-OR2
R3O-CH O O O
I I l I l I l
CH2- O— P— O— CH2CH2-N- C— (CH2) - C— XR1 I H
OH
( D
wherein X represents -O-, -NH- or -S-, R1 represents an alkyl group, a cycloalkyl group or an aryl group, R2 and R3 independently represent an acyl group, and m represents an integer of 1 to 10.
7. The lipid composition according to claim 6, wherein R2 and R3 independently represent a C 12 to C22 acyl group derived from a saturated or unsaturated fatty acid.
8. The lipid composition according to claim 6 or 7, wherein m is an integer of 2 to
4.
9. The lipid composition according to any one of claims 6 to 8, wherein X is -O- or -NH-, and R1 is an alkyl group or an aryl group.
10. The compound according to any one of claims 6 to 9, wherein X is -O-, and R1 is an alkyl group.
11. The lipid composition according to any one of claims 6 to 10, wherein other phospholipids are selected from phosphatidylcholine, phosphatidic acid, phosphatidylserine and phosphatidylglycerol.
12. The lipid composition according to any one of claims 6 to 11, which further comprises other neutral lipids.
13. The lipid composition according to claim 12, wherein other neutral lipids are selected from cholesterol and derivatives thereof.
14. The lipid composition according to claim 12 or 13, which further comprises a phosphatidylethanolamine dicarboxylic acid derivative represented by the following general formula (2), or salts thereof: [Chemical Formula 3]
H2C-OR4
R3O-CH O O O
II
C -HH22-- OO--P-O-CH2CH2-N-C-(CH2) — C-OH
OH
( 2 )
wherein R4 and R5 independently represent an acyl group, and n is an integer of 1 to 10.
15. The lipid composition according to claim 14, wherein R4 and R5 independently represent a C12 to C22 acyl group derived from a saturated or unsaturated fatty acid.
16. The lipid composition according to claim 14 or 15, wherein n is an integer of 2 to 4.
17. The lipid composition according to any one of claims 14 to 16, wherein an in vivo target factor is bound to at least a part of the phosphatidylethanolamine dicarboxylic acid derivative of the formula (2).
18. The lipid composition according to claim 17, wherein the in vivo target factor is selected from transferrin, folic acid, hyaluronic acid, a sugar chain, an antibody and an antibody fragment.
19. The lipid composition according to claim 18, wherein the in vivo target factor is transferrin.
20. A liposome comprising a phosphatidylethanolamine derivative represented by the following general formula (1), or salts thereof:
[Chemical Formula 4] H2C-OR2
R3O-CH O O O
CH2-O- P— O— CH2CH2- -NN-- C— (CH2) - C— XR1 H
OH
( D wherein X represents -O-, -NH- or -S-, R1 represents an alkyl group, a cycloalkyl group or an aryl group, R2 and R3 independently represent an acyl group, and m represents an integer of 1 to 10.
21. The liposome according to claim 20, wherein R2 and R3 independently represent a C 12 to C22 acyl group derived from a saturated or unsaturated fatty acid.
22. The liposome according to claim 20 or 21, wherein m is an integer of 2 to 4.
23. The liposome according to any one of claims 20 to 22, wherein X is -O- or -NH-, and R1 is an alkyl group or an aryl group.
24. The liposome according to any one of claims 20 to 23, wherein X is -O-, and R1 is an alkyl group.
25. The liposome according to any one of claims 20 to 24, which further comprises other phospholipid.
26. The liposome according to claim 25, wherein other phospholipids are selected from phosphatidylcholine, phosphatidic acid, phosphatidylserine and phosphatidylglycerol.
27. The liposome according to claim 25 or 26, which further comprises other neutral lipids.
28. The liposome according to claim 27, wherein other neutral lipids are selected from cholesterol and derivatives thereof.
29. The liposome according to claim 27 or 28, which further comprises a phosphatidylethanolamine dicarboxylic acid derivative represented by the following general formula (2), or salts thereof:
[Chemical Formula 5]
H2C-OR4
R5O-CH O O O
I Il I l I l
CH2-O-P-O-CH2CH2-N-C-(CH2) — C~0H I H
OH
( 2 )
wherein R4 and R5 independently represent an acyl group, and n is an integer of 1 to 10.
30. The liposome according to claim 29, wherein R4 and R independently represent a C 12 to C22 acyl group derived from a saturated or unsaturated fatty acid.
31. The liposome according to claim 29 or 30, wherein n is an integer of 2 to 4.
32. The liposome according to any one of claims 29 to 31, wherein an in vivo target factor is bound to at least a part of the phosphatidylethanolamine dicarboxylic acid derivative of the formula (2).
33. The liposome according to claim 32, wherein the in vivo target factor is selected from transferrin, folic acid, hyaluronic acid, a sugar chain, an antibody and an antibody fragment.
34. The liposome according to claim 33, wherein the in vivo target factor is transferrin.
35. The liposome according to any one of claims 20 to 34, which comprises a drug, a nucleic acid or an inert gas incorporated therein.
36. The liposome according to claim 35, wherein the drug is a medical drug or a diagnostic drug.
37. The liposome according to claim 36, wherein the medical drug is an antitumor substance.
PCT/US2007/077537 2006-09-05 2007-09-04 Phosphatidylethanolamine derivative and liposome containing the same WO2008030807A2 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190134226A1 (en) * 2012-08-01 2019-05-09 Nationwide Children's Hospital Intrathecal delivery of recombinant adeno-associated virus 9
CN113307824A (en) * 2021-04-26 2021-08-27 浙江大学 Amphiphilic material and application thereof in preparation of liposome

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4751219A (en) * 1985-02-05 1988-06-14 Nederlandse Centrale Organisatie Voor Toegepast-Natuur-Wetenschappelijk Onderzoek Synthetic glycolipides, a process for the preparation thereof and several uses for these synthetic glycolipides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4751219A (en) * 1985-02-05 1988-06-14 Nederlandse Centrale Organisatie Voor Toegepast-Natuur-Wetenschappelijk Onderzoek Synthetic glycolipides, a process for the preparation thereof and several uses for these synthetic glycolipides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190134226A1 (en) * 2012-08-01 2019-05-09 Nationwide Children's Hospital Intrathecal delivery of recombinant adeno-associated virus 9
CN113307824A (en) * 2021-04-26 2021-08-27 浙江大学 Amphiphilic material and application thereof in preparation of liposome
WO2022228230A1 (en) * 2021-04-26 2022-11-03 浙江大学 Amphiphilic material and application thereof in preparation for liposome

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