Phospholipid derivatives and process for the production there

专利摘要:
A phospholipid derivative represented by the following formula (1):wherein [PG]k represents a residue of polyglycerin having a polymerization degree of k,wherein k is 2 to 50, R1CO and R2CO independently represent an acyl group having 8to 22 carbon atoms, symbol "a" independently represents an integer of 0 to 5, symbol"b" independently represents 0 or 1, M represents hydrogen atom, an alkali metal atom,an ammonium, or an organic ammonium, and k1, k2, and k3 represent numberssatisfying the following conditions: 1 ≦ k1 ≦ (k+2)/2, 0 ≦ k2, and k1 + k2 + k3 = k+ 2. The phospholipid derivative is highly safe for living bodies and can be suitablyutilized in drug delivery systems such as liposome, and the like.
公开号:EP1591447A1
申请号:EP03778894
申请日:2003-12-12
公开日:2005-11-02
发明作者:Kazuhiro Kubo；Chika Itoh；Syunsuke Ohhashi；Tohru Yasukohchi；Yusuke Ohkawa；Hiroshi DAIICHI PHARMACEUTICAL CO. LTD. KIKUCHI；Norio Daiichi Pharmaceutical Co. Ltd. SUZUKI；Miho DAIICHI PHARMACEUTICAL CO. LTD. TAKAHASHI；Hitoshi DAIICHI PHARMACEUTICAL CO. LTD YAMAUCHI
申请人:Daiichi Seiyaku Co Ltd；NOF Corp；
IPC主号:A61K9-00

专利说明:
Technical Field
The present invention relates to a phospholipid derivative containingpolyglycerin and a method for producing the same. The present invention also relatesto a surfactant, solubilizer, dispersing agent for cosmetics and lipid membranestructure containing the phospholipid derivative.Background Art
Microparticle drug carriers including liposomal drug as typical examples andpolypeptides such as protein drug are known to have poor retention in blood and beeasily captured by the reticuloendothelial system (hereinafter abbreviated as "RES")such as liver and spleen when they are intravenously administered. The presence ofRES is a serious obstacle when a microparticle drug carrier is utilized as a targetingtype preparation, which delivers a medicament to organs other than RES, and as asustained-release preparation, which allows a medicament retained in blood for a longperiod of time to control the release of the medicament.
Researches have so far been conducted to impart a microcirculation propertyto the aforementioned preparations. Some proposals have been made, including, forexample, a method of maintaining a high blood concentration by reducing a size ofliposomes in view of relative easiness of a control of physicochemical properties of lipidbilayers of liposomes (Biochimica et BiophysicaActa, Vol. 761, p.142, 1983), a methodof utilizing lecithin having a high phase transfer temperature (BiochemicalPharmacology, Vol. 32, p. 3381, 1983), a method of utilizing sphingomyelin instead oflecithin (Biochemical Pharmacology, Vol. 32, p. 3381, 1983), a method of addingcholesterol as a membrane component of liposomes (Biochimica et Biophysica Acta, Vol.761, p. 142, 1983) and the like. However, by applying the aforementioned method, nowork has been known so far that successfully provides a microparticle drug carrierhaving favorable retention in blood and being hardly taken up by RES.
As another approach for solution, researches have been made for providing a microcirculation property and escapability from RES by modification of membranesurfaces of liposomes with a glycolipid, glycoprotein, amino acid-lipid, polyethyleneglycol-lipid or the like. Substances for the modification so far reported include, forexample, glycophon (The Pharmaceutical Society of Japan, the 106th Annual Meeting,Summaries of Symposia, p.336, 1986), ganglioside GM1 (FEBS Letters, Vol. 223, p.42,1987), phosphatidylinositol (FEBS Letters, Vol. 223, p.42, 1987), glycophon andganglioside GM3 (Japanese Patent Unexamined Publication (Kokai) No. 63-221837),polyethylene glycol derivative (FEBS Letters, Vol. 268, p.235, 1990), glucuronic acidderivative (Chemical & Pharmaceutical Bulletin, Vol. 38, p.1663, 1990), glutamic acidderivative (Biochimica et Biophysica Acta, Vol. 1108, p.257, 1992), polyglycerinphospholipid derivative (Japanese Patent Unexamined Publication No. 6-228012), andthe like.
As the modification of a polypeptide, introduction of two water-solublepolymer molecules into a polypeptide by using triazine has been reported for a purposeof decreasing the number of binding sites of the polypeptide and thereby increasing aresidual amount of active groups such as lysine residues in the polypeptide. Also asfor a liposome preparation, introduction of two water-soluble polymer molecules intotriazine to increase the molecular weight of the water-soluble polymer, andmodification of liposome surfaces by using the resulting polymer is reported.However, when a water-soluble polymer is introduced by using triazine, only twowater-soluble polymers can be introduced into the triazine ring. Therefore, it isnecessary to add a large amount of a compound, which contains two water-solublepolymers introduced in triazine, to increase the number of the water-soluble polymerchains on liposome surfaces. Further, a compound consisting of two or threepolyalkylene glycol chains bonded with one functional group has been reported as apolymer modifier. However, the number of the polymer chains, for which thismodification can be applied, is limited to 2 or 3, and the aforementioned compoundcannot have more than one functional group, because the ends of the polyalkyleneglycol chains, except for one end, are blocked with methyl group or ethyl group. It isexpected that the effect of this compound to impart microcirculation property toliposome surfaces is inferior to that of a compound having a hydrophilic group.Furthermore, although phospholipid derivatives containing a polyalkylene oxide group have also been used also as surfactants, no compound has been known so far that issafe for living bodies and can be stably used under a condition of a high saltconcentration.Disclosure of the Invention
An object of the present invention is to provide a phospholipid derivative thatis safe for living bodies and can be suitably used in the fields of solubilization anddispersion of physiologically active substances and the like, drug delivery systems suchas liposomes, and cosmetics. The inventors of the present invention conductedvarious researches to achieve the aforementioned object. As a result, they found thatnovel phospholipid derivatives containing a polyglycerin represented by the followingformula had the desired properties. The present invention was achieved on the basisof these findings.
The present invention thus provides a phospholipid derivative, which isrepresented by the following formula (1):
wherein [PG]k represents a residue of polyglycerin having a polymerization degree of k,wherein k is 2 to 50, R¹CO and R²CO independently represent an acyl group having 8to 22 carbon atoms, symbol "a" independently represents an integer of 0 to 5, symbol"b" independently represents 0 or 1, M represents hydrogen atom, an alkali metal atom,an ammonium, or an organic ammonium, and k1, k2, and k3 represent numberssatisfying the following conditions: 1 ≦ k1 ≦ (k+2)/2, 0 ≦ k2, and k1 + k2 + k3 = k+ 2.
According to preferred embodiments, the present invention provides the aforementioned phospholipid derivative represented by the aforementioned formula (1),wherein k1 satisfies 1≦ k1 ≦2; the aforementioned phospholipid derivativerepresented by the aforementioned formula (1), wherein k2 satisfies 0 ≦ k2 ≦ 1; theaforementioned phospholipid derivative represented by the aforementioned formula (1),wherein k1, k2, and k3 satisfy 8 ≦ k1 + k2 + k3 ≦ 52; the aforementionedphospholipid derivative represented by the aforementioned formula (1), wherein R¹COand R²CO independently represent an acyl group having 12 to 20 carbon atoms; theaforementioned phospholipid derivative represented by the aforementioned formula (1),wherein k2 is 0; the aforementioned phospholipid derivative represented by theaforementioned formula (1), wherein a and b represent 0; and the aforementionedphospholipid derivative represented by the aforementioned formula (1), wherein k2satisfies 0 < k2.
From other aspects, the present invention provides a surfactant comprisingthe aforementioned phospholipid derivative represented by the aforementionedformula (1); a solubilizer comprising the aforementioned phospholipid derivativerepresented by the aforementioned formula (1); a dispersing agent, preferably adispersing agent for cosmetics, comprising the aforementioned phospholipid derivativerepresented by the aforementioned formula (1); and a lipid membrane structure,preferably a liposome, containing the aforementioned phospholipid derivativerepresented by the aforementioned formula (1).
From a further aspect, the present invention provides a method for producingthe aforementioned phospholipid derivative represented by the aforementionedformula (1), which comprises the step of reacting a compound represented by thefollowing formula (2):
wherein R¹, R², a, and M have the same meanings as those defined above, and Xrepresents hydrogen atom or N-hydroxysuccinimide, and a polyglycerin represented by the following formula (3):
wherein [PG]k represents a residue of polyglycerin having a polymerization degree of k,wherein k has the same meaning as that defined above, and k4 is a number satisfyingthe following condition: k4 = k + 2. This method can be preferably performed in anorganic solvent in the presence of a basic catalyst, more preferably at a temperaturewithin the range of 20 to 90°C in the presence of a dehydration condensation agent.
The present invention also provides a method for producing a phospholipidderivative represented by the formula (1), which comprises the following steps: (A) the step of reacting a polyglycerin and a dibasic acid or a halogenated carboxylicacid to obtain a carboxylated polyglycerin; and (B) the step of reacting the carboxylated polyglycerin obtained in the aforementionedstep (A) and a phospholipid, and a method for producing a phospholipid derivativerepresented by the formula (1), which comprises the following steps: (A') the step of reacting a polyglycerin and a halogenated carboxylic acid ester andhydrolyzing the obtained ester compound to obtain a carboxylated polyglycerin; and (B) the step of reacting the carboxylated polyglycerin obtained in the aforementionedstep (A) and a phospholipid.

The present invention further provides a method for producing a phospholipidderivative represented by the formula (1) (except for a compound wherein k2 is 0),which comprises the step of reacting a polyglycerin derivative represented by thefollowing formula (4):
wherein [PG]k represents a residue of polyglycerin having a polymerization degree ofk, wherein k represent a number of 2 to 50, Y represents hydroxyl group or a leavinggroup, and k5 and k6 are numbers satisfying the following conditions: 1 ≦ k5 ≦(k+2)/2, and k5 + k6 = k + 2, and a phospholipid represented by the following formula(5):
wherein R¹ and R² have the same meanings as those defined above. This method canbe preferably performed in an organic solvent in the presence of a basic catalyst, morepreferably at a temperature within the range of 20 to 90°C.
From a still further aspect, the present invention provides a pharmaceuticalcomposition comprising a lipid membrane structure (preferably liposome) containingthe phospholipid derivative represented by the aforementioned formula (1) andretaining a medicament. The aforementioned pharmaceutical composition whereinthe medicament is an antitumor agent is provided as a preferred embodiment.Best Mode for Carrying out the Invention
In the phospholipid derivative of the present invention represented by theformula (1), [PG]k represents a residue of polyglycerin having a polymerization degreeof k, and k1 + k2 + k3 is k + 2. Symbol "k" represents a polymerization degree , andgenerally means an average polymerization degree. The residue of polyglycerinmeans a remaining portion of the polyglycerin excluding all of the hydroxyl groups.The polyglycerin constituting the phospholipid derivative represented by the formula(1) is a compound consisting of two or more glycerin molecules linked via ether bonds.For example, when the polyglycerin exists as a linear chain compound, the compoundis represented by the formula:HO-CH₂-CH(OH)-CH₂-[O-CH₂-CH(OH)-CH₂]_k-2-O-CH₂-CH(OH)-CH₂-OH(k is an integer of 2 or more, and means the number of glycerin molecules involved in the polymerization (also sometimes referred to as "polymerization degree")). It can bereadily understood by those skilled in the art that the polyglycerin can exist as abranched chain compound. Therefore, the term of polyglycerin used in thespecification should not be construed in any limitative way to mean only a linear chaincompound. Specific examples of the polyglycerin include diglycerin, triglycerin,tetraglycerin, pentaglycerin, hexaglycerine, heptaglycerin, octaglycerin, nonaglycerin,decaglycerin, didecaglycerin, tridecaglycerin, tetradecaglycerin, and the like. Asingle substance may be used as the polyglycerin. Alternatively, a mixture of two ormore kinds of linear chain and/or branched chain polyglycerin residues having thesame or similar polymerization degrees can also be used, and a compound having theresidue of polyglycerin such as mentioned above also falls within the scope of thepresent invention.
Symbol "k1" means the number of residues of the phospholipid compoundbonded to the residue of polyglycerin, and the number is 1 to (k+2)/2. When thenumber of the bonding residues of phospholipid compound k1 is less than 1, theadvantageous effects of the present invention cannot be obtained due to smallernumbers of hydrophobic bond portions in a molecule. Further, when the compound ofthe present invention is used for a lipid membrane structure, k1 preferably satisfiesthe condition of 1 ≦ k1 ≦ 2. When the number of the bonding residues ofphospholipid compound satisfies the condition of 2 < k1 ≦ (k+2)/2, namely, when k1 ismore than 2, the residues of the phospholipid compound contained in the compound ofthe present invention increase, in other words, a lot of hydrophobic portions exist inthe molecule. Therefore, the compound becomes more likely to form micelles, andthus the compound can be suitably used as a solubilizer or a dispersing agent.
Symbol "k2" represents the number of groups that bond to the residue ofpolyglycerin of which end is represented by -COOM, and k2 satisfies the condition of 0≦ k2. When k2 is 0, it means that any partial structure, of which end is representedby -COOM, does not substantially exist in the compound of the present invention.Further, when k2 is more than 0, carboxyl groups exist and as a result the compoundhas polarity. Therefore, the compound can be used for a dispersing agent and the likeas an ionic surfactant. When k2 satisfies the condition of 0 ≦ k2 ≦ 1, the compounddoes not unstabilize a lipid membrane structure such as liposome, but can stabilize liposomes due to a small number of carboxyl groups, and therefore the compound canbe preferably used. M represents hydrogen atom, an alkali metal atom, anammonium, or an organic ammonium, preferably hydrogen atom or an alkali metalatom. Specific examples include, for example, an alkali metal atom such as sodiumand potassium, an organic ammonium such as triethylammonium anddiisopropylammonium, and the like.
Symbol "k3" is the number of the hydroxyl groups that bond to thepolyglycerin residue, and the number is an integer satisfying the condition of k1 + k2 +k3 = k + 2. The value of k1 + k2 + k3 is an integer of 4 to 52, preferably 8 to 52, morepreferably 8 to 12. When the value of k1 + k2 + k3 is smaller than 4, theadvantageous effects of the present invention may not be fully obtained. When thevalue of k1 + k2 + k3 is larger than 52, viscosity of the polyglycerin becomes large, andit may become difficult to obtain such a compound.
R¹CO and R²CO independently represent an acyl group having 8 to 24 carbonatoms, preferably 12 to 20 carbon atoms. The type of the acyl group is notparticularly limited, and either an aliphatic acyl group or an aromatic acyl group maybe used. However, in general, an acyl group derived from a fatty acid can bepreferably used. Specific examples of R¹CO and R²CO include an acyl group derivedfrom a saturated or unsaturated linear or branched fatty acid such as caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid,isostearic acid, oleic acid, linoleic acid, arachic acid, behenic acid, erucic acid, andlignoceric acid. R¹CO and R²CO may be the same or different. When the number ofcarbon atoms exceeds 24, reactivity may sometimes be degraded due to poor dispersionin an aqueous phase. When the number of carbon atoms is less than 8, final purity ofthe objective substance may sometimes be degraded due to poor crystallizing propertyduring a purification process.
In the formula (1), symbol "b" is independently an integer of 0 or 1. When b is1, it is preferred that symbol "a" is an integer of 1 to 4, more preferably 2 or 3. When bis 0, it is preferred that a is 0.
Although the method for producing the compound of the present inventionrepresented by the formula (1) is not particularly limited, the compound can beconveniently produced by any of the following methods depending on the structure of the target compound.<Production Method A>
The phospholipid derivative wherein k2 is 0 can be produced with high purityby, for example, reacting a compound represented by the formula (2) with a compoundrepresented by the formula (3). In the phospholipid compound represented by theformula (2), R¹, R², M, and a are the same as those explained for the formula (1), and Xis hydrogen atom or N-hydroxysuccinimide.
The phospholipid compound represented by the formula (2) used as a rawmaterial can be produced by a known method. For example, the compound can beeasily produced by reacting a phospholipid compound with a dicarboxylic acidanhydride. The phospholipid to be used may be a natural phospholipid or syntheticphospholipid so long as a phospholipid satisfying the definitions of R¹ and R² is chosen.Examples include, for example, natural and synthetic phosphatidylethanolaminessuch as soybean phosphatidyldiethanolamine and hydrogenated soybeanphosphatidyldiethanolamine, yolk phosphatidyldiethanolamine and hydrogenated yolkphosphatidyldiethanolamine, and the like.
The compound of the present invention represented by the formula (1) can alsobe produced by reacting an activated ester derivative of a phospholipid compoundrepresented by the formula (2) with a polyglycerin compound represented by theformula (3). The aforementioned activated ester derivative can be obtained by, forexample, reacting a phospholipid compound represented by the formula (2) wherein Xis hydrogen atom with an activator in the presence of a dehydration condensationagent. The type of the aforementioned activator is not particularly limited, andexamples include, for example, N-hydroxysuccinimide, N,N' -disuccinimide carbonate,1-hydroxybenzotriazole, 4-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide,N-hydroxyphthalimide, 4-hydroxyphenyldimethylsulfonium/methyl sulfate, and thelike. Among them, N-hydroxysuccinimide is preferred.
The reaction of the phospholipid compound represented by the formula (2) andthe activator can be performed in a solvent that does not react with a carboxylic acidsuch as chloroform and toluene at a reaction temperature of 15 to 80°C, preferably 25to 55°C, in the presence of a dehydration condensation agent, and the reaction can beperformed by, for example, dispersing the activator in a solution of the phospholipid compound with stirring. For example, when N-hydroxysuccinimide is used as theactivator, the carboxyl group of the phospholipid compound represented by the formula(2) and the imide group of N-hydroxysuceinimide will react to produce an activatedester derivative wherein N-hydroxysuccinimide binds to the end of the phospholipidcompound represented by the formula (2) on the side of the carboxyl group.
As the organic solvent used for the reaction, those having no reactivefunctional group such as hydroxyl group can be used without particular limitation.Examples include, for example, ethyl acetate, dichloromethane, chloroform, benzene,toluene, and the like. Among them, chloroform and toluene are preferred. Organicsolvents having hydroxyl group such as ethanol may react with the carboxyl group atthe end of the polyglycerin compound represented by the formula (4).
The reaction of the phospholipid compound represented by the formula (2) andthe polyglycerin compound represented by the formula (3) can be usually performed inan organic solvent in the presence of a basic catalyst, and the reaction can bepreferably performed by using a dehydration condensation agent. The type of thebasic catalyst is not particularly limited, and examples include, for example,nitrogen-containing substances such as triethylamine, pyridine,dimethylaminopyridine, and ammonium acetate, organic salts such as sodiumphosphate, sodium carbonate, sodium hydrogencarbonate, sodium borate, and sodiumacetate, and the like. The amount of the basic catalyst may be a minimum amount tocomplete the reaction, considering the purification step and the like. The basiccatalyst is desirably used generally in an amount of 1 to 2 moles, preferably 1 to 1.5moles, per mole of the phospholipid compound represented by the formula (2), if areaction rate with the phospholipid compound represented by the formula (2) is takeninto consideration. As the organic solvent, those having no reactive functional groupsuch as hydroxyl group can be used without particular limitation. Examples include,for example, ethyl acetate, dichloromethane, chloroform, dimethyl sulfoxide, benzene,toluene, and the like. Among them, dimethyl sulfoxide, chloroform, and toluene arepreferred. Organic solvents having hydroxyl group such as ethanol may react withthe carboxyl group at the end of the phospholipid compound represented by theformula (2).
When a dehydration condensation agent is used, the type of the dehydration condensation agent is not particularly limited so long as the agent can achievedehydration condensation of the polyglycerin compound represented by the formula (3)and a functional group of the phospholipid compound represented by the formula (2).Examples of the dehydration condensation agent include, for example, carbodiimidederivatives such as dicyclohexylcarbodiimide and diisopropylcarbodiimide, anddicyclohexylcarbodiimide is especially preferred. The amount of the dehydrationcondensation agent used is not particularly limited. However, the polyglycerincompound represented by the formula (3) has many hydroxyl groups, and as a result,has hygroscopic property and contains a lot of moisture. Accordingly, carbodiimidederivatives such as dicyclohexylcarbodiimide and diisopropylcarbodiimide may reactwith the moisture in the polyglycerin, and thus the objective dehydration condensationreaction of the polyglycerin compound represented by the formula (3) and thefunctional group of the phospholipid compound represented by the formula (2) maypossibly not be completed. Therefore, the amount of the dehydration condensationagent is, for example, preferably about 1 to 10 moles, more preferably about 1 to 5moles, per mole of the phospholipid compound represented by the formula (2).
By addition of N-hydroxysuccinimide to the reaction system in an amount of0.1 to 2 moles per mole of the phospholipid compound represented by the formula (2), areaction rate can be increased.
The amount of the phospholipid compound represented by the formula (2) isnot particularly limited. The amount is preferably 1 to 3 moles, more preferably 1 to1.3 moles based on the number of k1 per one molecule.
The reaction temperature is usually 20 to 90°C, preferably 40 to 80°C. Thereaction time is 1 hour or longer, preferably 2 to 8 hours. When the reactiontemperature is lower than 20°C, the reaction rate may sometimes be low. When thereaction temperature is higher than 90°C, the acyl group in the phospholipid compoundrepresented by the formula (2) used for the reaction may sometimes be hydrolyzed. Inaddition, although the compound of the present invention may be obtained as a singlecompound depending on a synthetic method, the compound may also be obtained as amixture of substances having different numbers for each of k1, k2, and k3. Such amixture also falls within the scope of the present invention. Further, the polyglycerinused as a raw material may sometimes not be a single substance, but is a mixture of polyglycerin compounds having two or more kinds of straight and/or branchedpolyglycerin residues and having the same or similar polymerization degrees. Whensuch material is used, the target substance may be obtained as a mixture of compoundshaving two or more kinds of structures as for the polyglycerin residue, which mixturealso falls within the scope of the present invention. This explanation shall also applyto the reaction steps explained below.<Production Method B>
The phospholipid derivative of the formula (1) wherein k2 is 0 and thephospholipid derivative of the formula (1) wherein k2 is not 0, i.e., the compoundwherein a polyglycerin residue is bonded with a partial structure having carboxylgroup at an end, can be produced by reacting a carboxylated polyglycerin with aphospholipid compound according to a method including the aforementioned steps (A)and (B). By reacting the polyglycerin compound with a dibasic acid or a halogenatedcarboxylic acid in the step (A) to obtain a carboxylated polyglycerin and then reactingthe resulting carboxylated polyglycerin with the phospholipid in the step (B), thecompound of the present invention can be easily obtained. In the step (A'), by reactinga halogenated carboxylic acid ester instead of the dibasic acid or halogenatedcarboxylic acid and then performing hydrolyzation, a carboxylated polyglycerin canalso be obtained.
Specific examples of the dibasic acid, halogenated carboxylic acid, andhalogenated carboxylic acid ester include succinic anhydride, glutaric anhydride,chloropropionic acid, methyl chloropropionate, ethyl chloropropionate, bromopropionicacid, methyl bromopropionate, ethyl bromopropionate, bromohexanoic acid, methylbromohexanoate, ethyl bromohexanoate, and the like. However, the dibasic acid,halogenated carboxylic acid and halogenated carboxylic acid ester to be reacted withthe polyglycerin compound are not limited to the aforementioned compounds, and anycompounds may be used so long as a compound successfully provides a carboxylatedpolyglycerin. The amount of the dibasic acid, halogenated carboxylic acid, orhalogenated carboxylic acid ester used in the step (A) or (A') is not particularly limited.The compounds are preferably added in a slightly excessive amount considering areaction rate. The amount is 1 to 2 moles, preferably 1 to 1.5 moles, based on adesired number of carboxyl groups determined by k2.
As the organic solvent used in the step (A) or (A'), those having no functionalgroup such as hydroxyl group can be used without particular limitation. Examplesinclude, for example, ethyl acetate, dichloromethane, chloroform, dimethyl sulfoxide,benzene, toluene, and the like. Among them, dimethyl sulfoxide, chloroform, andtoluene are preferred. Organic solvents having hydroxyl group such as ethanol willreact with the dibasic acid, halogenated carboxylic acid and halogenated carboxylicacid ester compound to be reacted with the polyglycerin, and therefore they are notpreferred. Although dichloromethane and the like do not have a problem concerningreactivity, they may not be practically preferred due to a low boiling point. A reactiontemperature of the step (A) or (A') is not particularly limited. The temperature maybe, for example, 20 to 110°C, preferably 30 to 90°C. A reaction time is not particularlylimited either, and may desirably be, for example, 1 hour or more, preferably 2 to 48hours. A reaction temperature below 20°C may not be preferred from a viewpoint ofreaction efficiency.
The phospholipid used in the step (B) may be a natural phospholipid orsynthetic phospholipid. Examples include, for example, natural and syntheticphosphatidylethanolamines such as soybean phosphatidyldiethanolamine andhydrogenated soybean phosphatidyldiethanolamine, yolk phosphatidyldiethanolamineand hydrogenated yolk phosphatidyldiethanolamine, and the like. As the organicsolvent used in the step (B), those having no functional group such as hydroxyl groupcan be used without particular limitation. Examples include, for example, ethylacetate, dichloromethane, chloroform, dimethyl sulfoxide, benzene, toluene, and thelike. Among them, dimethyl sulfoxide, chloroform, and toluene are preferred.Organic solvents having hydroxyl group such as ethanol will react with the dibasicacid, halogenated carboxylic acid and halogenated carboxylic acid ester compound to bereacted with the polyglycerin, and therefore they are not preferred. Althoughdichloromethane and the like do not have a problem concerning reactivity, they maynot be practically preferred due to a low boiling point. A reaction temperature of thestep (B) is not particularly limited, and may be, for example, 20 to 100°C, preferably 20to 90°C. A reaction time is not particularly limited either, and may desirably be, forexample, 0.5 to 24 hours, preferably 1 to 12 hours. A reaction temperature below 20°Cmay not be preferred from a viewpoint of reaction efficiency.
For the reaction of the phospholipid compound and carboxylated polyglycerinperformed in the step (B), a dehydration condensation agent and/or a basic catalyst canbe used. As the dehydration condensation agent, those allowing dehydrationcondensation of the carboxyl group of the carboxylated polyglycerin and a functionalgroup of the phospholipid compound can be used without particular limitation.Examples of the dehydration condensation agents include, for example, carbodiimidederivatives such as dicyclohexylcarbodiimide. As the dehydration condensation agent,dicyclohexylcarbodiimide is preferred. An amount of the dehydration condensationagent used is desirably about 1 to 5 moles, more preferably about 1 to 2 moles, per moleof the phospholipid compound. Further, it is preferable to add N-hydroxysuccinimideto the reaction system in an amount of 0.1 to 2 moles per mole of the phospholipidcompound to increase the reaction efficiency. The type of the basic catalyst used forthis reaction is not particularly limited, and examples include, for example,nitrogen-containing substances such as triethylamine, dimethylaminopyridine, andammonium acetate, organic salts such as sodium phosphate, sodium carbonate, sodiumhydrogencarbonate, sodium borate, and sodium acetate, and the like. An amount ofthe basic catalyst is not particularly limited, and may be, for example, 1 to 5 moles,preferably 1 to 2 moles, per mole of the phospholipid compound used in the step (B).An amount of the phospholipid compound used in the step (B) is not particularlylimited, and the compound can be suitably reacted depending on a desired number ofk1. For example, the amount is preferably 1 to 3 moles, more preferably 1 to 1.3moles, based on the number of k1 per one molecule.<Production Method C>
As for the polyglycerin-modified phospholipid of the present invention, thephospholipid derivative of the formula (1) wherein k2 is 0, and the phospholipidderivative of the formula (1) wherein k2 is not 0, and a and b are 0 can be easilysynthesized by reacting a polyglycerin compound represented by the formula (4) with aphospholipid represented by the formula (5). In the polyglycerin compoundrepresented by the formula (4), [PG]k represents a residue of polyglycerin having apolymerization degree of k, wherein k represent a number of 2 to 50, Y representshydroxyl group or a leaving group, and k5 and k6 are numbers satisfying the followingconditions: 1 ≦ k5 ≦ (k+2)/2, and k5 + k6 = k + 2. In the polyglycerin compound represented by the formula (4), Y represents hydroxyl group or a leaving group. Inthe specification, the "leaving group" is a group which imparts to the polyglycerincompound reactivity with a phospholipid, and includes electron withdrawing groupsand other groups. Specifically, examples of such a group include imidazole group,4-nitrophenyloxy group, benzotriazole group, chlorine, methoxy group, ethoxy group,propyloxy group, carbonyloxcy-N-2-pyrrolidinone group, carbonyl- 2-oxypyrimidinegroup, N-succinimidyloxy group, pentafluorobenzoyl group, and the like. Amongthem, imidazole group, 4-nitrophenyloxy group, benzotriazole group, chlorine, and N-succinimidyloxygroup are preferred, and N-succinimidyloxy group and4-nitrophenyloxy group are particularly preferred.
Examples of the method for obtaining the polyglycerin compound representedby the formula (4) include, for example, a method of introducing the aforementionedleaving group into the polyglycerin compound by using an activating agent such asN,N'-succinimidyl carbonate and chloroformic acid p-nitrophenyl ester in an organicsolvent in the presence of a basic catalyst such as triethylamine ordimethylaminopyridine, and the like. However, the method is not limited to theabove method, and the polyglycerin compound represented by the formula (4) may beproduced by any kind of method. An amount of the activating agent may generally beequimolar or more of k1 as being the number of the phospholipid to be introduced.However, the amount may preferably be 1 to 2 moles based on the number of k1substantially considering a purity of the activating agent and the like.
The phospholipid, which is used to synthesize the compound of the presentinvention represented by the formula (1) wherein a and b are 0 by using thepolyglycerin compound represented by the formula (4), is represented by the formula(5). This phospholipid may be a natural phospholipid or synthetic phospholipid.Examples include, for example, natural and synthetic phosphatidylethanolaminessuch as soybean phosphatidyldiethanolamine and hydrogenated soybeanphosphatidyldiethanolamine, yolk phosphatidyldiethanolamine and hydrogenated yolkphosphatidyldiethanolamine, and the like. A basic catalyst can be used for thisreaction, and the type of the basic catalyst is not particularly limited. Examplesinclude, for example, nitrogen-containing substances such as triethylamine,dimethylaminopyridine, and ammonium acetate, organic salts such as sodium phosphate, sodium carbonate, sodium hydrogencarbonate, sodium borate, and sodiumacetate, and the like. An amount of the basic catalyst is not particularly limited, andmay be, for example, 1 to 5 moles, preferably 1 to 2 moles, per mole of the phospholipidcompound used in the step (B). An amount of the phospholipid compound used in thestep (B) is not particularly limited, and can be suitably reacted depending on theobjective number of k1. For example, the amount may preferably be 1 to 3 moles,more preferably 1 to 1.3 moles based on the number of k1 for one molecule.
As the organic solvent used for this reaction, those having no functional groupsuch as hydroxyl group can be used without particular limitation. Examples include,for example, ethyl acetate, dichloromethane, chloroform, benzene, dimethyl sulfoxide(DMSO), toluene, and the like. Among them, chloroform, DMSO, and toluene arepreferred. Organic solvents having hydroxyl group such as ethanol will react with theleaving group at the end of the polyglycerin compound represented by the formula (4),and therefore they are not preferred. Although dichloromethane and the like do nothave a problem concerning reactivity, they may not be practically preferred due to alow boiling point. A reaction temperature of this reaction is not particularly limited,and may be, for example, 20 to 110°C, preferably 30 to 90°C. A reaction time is notparticularly limited and may desirably be, for example, 1 hour or more, preferably 2 to24 hours. A reaction temperature below 20°C may not be preferred from a viewpointof reaction efficiency, and at a reaction temperature higher than 90°C, the acyl group ofthe phospholipid compound used for the reaction may be hydrolyzed.
By using the compound of the present invention represented by theaforementioned formula (1) as a surfactant, a solubilized solution, emulsion, anddispersion can be obtained. The compound of the present invention is particularlyuseful as a solubilizer, emulsifier, or dispersing agent for hardly water-solublemedicaments. When the surfactant of the present invention is used as an emulsifier,solubilizer, or dispersing agent, the emulsifier, solubilizer, or dispersing agent maysolely contain the surfactant of the present invention, or may also contain other knowncomponents used for emulsification, solubilization, or dispersion. The form of thesolubilized solution or dispersion is not limited, and examples include a solution inwhich a fat-soluble substance or the like is dissolved in a dispersion medium such aswater and a buffer, or a dispersion in which a fat-soluble substance or the like is dispersed in a dispersion medium such as water and a buffer and the like.
Formulation of the emulsion and solubilized solution are not limited, andexamples include a micelle solution formed with the surfactant of the presentinvention, i.e., a micelle solution in which micelles contain a fat-soluble substance inthe inside thereof, an emulsion in which dispersed particles formed with the surfactantof the present invention and a fat-soluble substance or the like exist as colloidalparticles or larger particles, and the like. Examples of the micelle solution includepolymer micelle solutions in which dispersed particles have a diameter of 10 to 300 nm.The emulsion may be of O/W type or W/O/W type. The fat-soluble substance that canbe solubilized or emulsified is not particularly limited, and examples thereof include ahigher alcohol, ester oil, triglycerin, tocopherol, higher fatty acid, hardly water-solublemedicaments, and the like.
The hardly water-soluble medicaments to be solubilized according to thepresent invention are not particularly limited, and those having a solubility of 1,000ppm or less in water at 25°C, those having a solubility of 10 mg/mL or less and the likeare used, for example. Examples of the hardly water-soluble medicaments include,for example, cyclosporin, amphotericin B, indomethacin, nifedipine, tacrolimus,melphalan, ifosfamide, streptozocin (streptozotocin), methotrexate, fluorouracil,cytarabine, tegafur, idoxido, paclitaxel, docetaxel, daunorubicin, bleomycin,medroxyprogesterone, phenofibrate, and the like.
The use as a dispersing agent in the field of cosmetics is also not particularlylimited. For example, when a water-soluble substance such as ascorbic acid isretained in an internal aqueous phase of a lipid membrane structure, a fat-solublesubstance such as tocopherol is retained in a lipid bilayer or the like, the objectivesubstance can be more stably dispersed in an aqueous solution by using the compoundof the present invention as a lipid membrane structure formulating agent. When thecompound is used as a surfactant or a dispersing agent, the amount of the compound ofthe present invention to be added is 0.1 to 20% by mass, preferably 0.5 to 7% by mass,more preferably 0.5 to 5% by mass, based on a total mass of an objective substance forsolubilization, dispersion, emulsification or the like.
Further, for solubilization of the hardly water-soluble medicament, an amountof the compound of the present invention varies depending on the solubility of the medicament and the like, and the amount may be decided depending on the solubility.Although the amount of the compound of the present invention is not limited to thefollowing amount, the amount may be, for example, 500 to 100,000 % by mass relativeto the total mass of an objective medicament.
The compounds of the aforementioned formula (1) wherein k2 is 0 can beespecially effectively used as a nonionic surfactant under a high salt concentrationcondition. Generally, polyglycerin-modified phospholipids and the like havehydrophilicity deriving from the glycerin group and hydrophobicity deriving from theacyl group, and therefore they can be used as surfactants. However, surfactantshaving oxyalkylene groups represented by polyalkylene oxide-modified phospholipidsgenerally have a problem in that they produce turbidity when they are used under ahigh salt concentration condition. In addition, the use of nonionic type surfactantsconsisting of glycidol derivatives under a high salt concentration condition has beenreported. However, such surfactants have a problem of skin irritation and the like,and thus have a problem of unsuitability for application in the cosmetic field. Thecompounds represented by the aforementioned formula (1) have a characteristicfeature in that they can maintain high solubilization ability even under a condition ofhigh salt concentration, and can be used as a surfactant having superior salt tolerance.Moreover, they can be used as a surfactant highly compatible with the skin in the fieldof cosmetics.
The compounds of the aforementioned formula (1) wherein k2 is more than 0,i.e., compounds having carboxyl group at the end of branched glycerin group, can beused as a pH sensitive phospholipid, for example, as a dispersing agent. When acationic substance (e.g., physiologically active cationic substance) or a basic substance,is dispersed in water, it can be stably dispersed in water by, for example, coating thesurfaces of microparticles or the like containing the cationic substance or basicsubstance with the aforementioned compound. The compound of the presentinvention has polyanionic groups, and thereby enables stable dispersion by ionicbonds.
The compounds of the present invention represented by the aforementionedformula (1) can be used as phospholipids constituting a lipid membrane structure suchas liposome, emulsion, and micelle. By using the compounds of the present invention, curculating time in blood of a lipid membrane structure, preferably liposome, can beincreased. This effect can be attained by adding a small amount of the compound ofthe present invention to a lipid membrane structure. Although it is not intended to bebound by any specific theory, it is considered that, when the compounds of the presentinvention having 4 or more of multiple branches are used as a phospholipidconstituting lipid membrane structure, the polyglycerin chains three-dimensionallyspread in the membranes of lipid membrane structure, and therefore aggregation ofmicroparticles in an aqueous solution is prevented to achieve a stable dispersion state.
The amount of the compound of the present invention added to a lipidmembrane structure may be an amount sufficient for effectively expressing efficacy ofa medicament in vivo and is not particularly limited. The amount can be suitablyselected depending on, for example, a type of medicament to be retained by the lipidmembrane structure, a purpose of therapeutic or prophylactic treatment and the like,and a form of the lipid membrane structure. A type of a medicament retained by thelipid membrane structure provided by the present invention is not particularly limited.For example, compounds used as antitumor agents are preferred. Examples of suchcompounds include, for example, camptothecin derivatives such as irinotecanhydrochloride, nogitecan hydrochloride, exatecan, RFS-2000, lurtotecan, BNP-1350,Bay-383441, PNU-166148, IDEC-132, BN-80915, DB-38, DB-81, DB-90, DB-91,CKD-620, T-0128, ST-1480, ST-1481, DRF-1042 and DE-310, taxane derivatives suchas docetaxel hydrate, paclitaxel, IND-5109, BMS-184476, BMS-188797, T-3782,TAX-1011, SB-RA-31012, SBT-1514 and DJ-927, ifosfamide, nimustine hydrochloride,carboquone, cyclophosphamide, dacarbazine, thiotepa, busulfan, melphalan,ranimustine, estramustine phosphate sodium, 6-mercaptopurine riboside, enocitabine,gemcitabine hydrochloride, carmofur, cytarabine, cytarabine ocphosphate, tegafur,doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine,fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicinhydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, doxorubicinhydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, zinostatinstimalamer, neocarzinostatin, mytomycin C, bleomycin sulfate, peplomycin sulfate,etoposide, vinorelbine tartrate, vincristine sulfate, vindesine sulfate, vinblastinesulfate, amrubicin hydrochloride, gefitinib, exemestan, capecitabine, TNP-470, TAK-165, KW-2401, KW-2170, KW-2871, KT-5555, KT-8391, TZT-1027, S-3304, CS-682,YM-511, YM-598, TAT-59, TAS-101, TAS-102, TA-106, FK-228, FK-317, E7070, E7389,KRN-700, KRN-5500, J-107088, HMN-214, SM-11355, ZD-0473 and the like.
Further, a gene or the like may be encapsulated in the lipid membranestructure of the present invention. The gene may be any of oligonucleotide, DNA, andRNA, and in particular, examples thereof include a gene for in vitro gene introductionsuch as transformation and a gene that act upon in vivo expression, for example, agene for gene therapy, gene used in breeding of industrial animals such as laboratoryanimals and livestock, and the like. Examples of the gene for gene therapy include anantisense oligonucleotide, antisense DNA, antisense RNA, gene coding for aphysiologically active substance such as enzymes and cytokines, and the like.
The aforementioned lipid membrane structure may further containphospholipids and a sterol such as cholesterol, and cholestanol, another fatty acidhaving a saturated or unsaturated acyl group having 8 to 24 carbon atoms and anantioxidant such as α -tocopherol. Examples of the phospholipid includephosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerin, cardiolipin, sphingomyelin, ceramidephosphorylethanolamine, ceramide phosphorylglycerin, ceramide phosphorylglycerinphosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, phosphatidicacid and the like, and they may be used alone or two or more kind of them can be usedin combination. The fatty acid residues of these phospholipids are not particularlylimited, and examples thereof include a saturated or unsaturated fatty acid residuehaving 12 to 20 carbon atoms. Specific examples include an acyl group derived from afatty acid such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid andlinoleic acid. Further, phospholipids derived from natural products such as egg yolklecithin and soybean lecithin can also be used.
The form of the lipid membrane structure of the present invention and thepreparation method thereof are not particularly limited, and examples of the existenceform thereof include, for example, a form of dried lipid mixture, form of dispersion inan aqueous solvent, dried or frozen form of the foregoing form and the like. The lipidmembrane structure in the form of dried lipid mixture can be prepared by, for example,first dissolving lipid components to be used in an organic solvent such as chloroform, and drying up the solution under reduced pressure by using an evaporator orspray-drying the solution by using a spray dryer. Examples of the form of the lipidmembrane structure dispersed in an aqueous solvent include unilamella liposomes,multilamella liposomes, O/W type emulsion, W/O/W type emulsion, spherical micelles,worm-like micelles, irregular layered structure and the like, and liposomes arepreferred among them. A size of the lipid membrane structure in the dispersed stateis not particularly limited. For example, the particle diameter of liposome or particlein emulsion is 50 nm to 5 µ m, and the particle diameter of spherical micelle is 5 to100 nm. When a worm-like micelle or irregular layered structure is formed, it can beconsidered that the thickness of one layer thereof is 5 to 10 nm, and such layers form asingle layer.
The composition of the aqueous solvent (dispersion medium) is also notparticularly limited, and the aqueous solvent may be, for example, a buffer such asphosphate buffer, citrate buffer, and phosphate-buffered physiological saline,physiological saline, a medium for cell culture or the like. The lipid membranestructure can be stably dispersed in these aqueous solvents. An aqueous solution of asugar such as glucose, lactose, and sucrose, an aqueous solution of a polyhydric alcoholsuch as glycerin and propylene glycol and the like may be further added. In order tostably store the lipid membrane structure dispersed in such an aqueous solvent for along period of time, it is desirable to minimize electrolytes in the aqueous solvent froma viewpoint of physical stability such as prevention of aggregation. Further, from aviewpoint of chemical stability of lipids, it is desirable to control a pH of the aqueoussolvent to be in a range of from weakly acidic pH to around neutral pH (pH 3.0 to 8.0),and to remove dissolved oxygen by nitrogen bubbling. Further, when a lyophilized orspray-dried product is stored, for example, use of an aqueous sugar solution or aqueouspolyhydric alcohol solution may enable effective storage at lyophilization and storageof an aqueous sugar solution. A concentration of these aqueous solvents is notparticularly limited. When an aqueous sugar solution is used, for example, theconcentration is preferably 2 to 20% (W/V), more preferably 5 to 10% (W/V), and whenan aqueous polyhydric alcohol solution is used, the concentration is preferably 1 to 5%(W/V), more preferably 2 to 2.5% (W/V). In a buffer, a concentration of the bufferingagent is preferably 5 to 50 mM, more preferably 10 to 20 mM. A concentration of the lipid membrane structure in an aqueous solvent is not particularly limited. Aconcentration of the total amount of lipids in the lipid membrane structure ispreferably 0.1 to 500 mM, more preferably 1 to 100 mM.
The formulation of the lipid membrane structure dispersed in an aqueoussolvent can be prepared by adding the aforementioned dried lipid mixture to anaqueous solvent and emulsifying the mixture by using an emulsifier such as ahomogenizer, ultrasonic emulsifier, high pressure jet emulsifier or the like. Further,the aforementioned form can also be prepared by a method known as a method forpreparing liposomes, for example, the reverse phase evaporation method, and themethod for preparing dispersion is not particularly limited. When it is desired tocontrol a size of the lipid membrane structure, extrusion (extrusion filtration) can beperformed under high pressure by using a membrane filter of even pore sizes or thelike.
Examples of the method for drying the aforementioned lipid membranestructure dispersed in an aqueous solvent include ordinary lyophilization and spraydrying. As the aqueous solvent used for these operations, an aqueous sugar solution,preferably aqueous sucrose solution or aqueous lactose solution, may be used asdescribed above. When a lipid membrane structure dispersed in the aqueous solventis first prepared and then successively dried, it becomes possible to store the lipidmembrane structure for a long period of time. In addition, when an aqueous solutionof a medicament is added to the dried lipid membrane structure, the lipid mixture isefficiently hydrated and thereby the medicament can be efficiently retained in the lipidmembrane structure, which provides an advantageous effect. For example, apharmaceutical composition can be prepared by adding a medicament to the lipidmembrane structure, and thus the lipid membrane structure can be used as apharmaceutical composition for therapeutic treatment and/or prevention of a disease.When the medicament is a gene, the composition can also be used as a gene deliverykit.
As for a formulation of the pharmaceutical composition, the formulation maybe the lipid membrane structures retaining a medicament, as well as a mixture of amedicament and the lipid membrane structures. The term "retain" used hereinmeans that a medicament exists inside the membranes of the lipid membrane structures, on the membrane surfaces, in the membranes, in the lipid layers, and/or onthe lipid layer surfaces. An available formulation of the pharmaceutical compositionand a method for preparation thereof are not particularly limited in the same manneras the lipid membrane structures. As for the available form, examples include a formof a dried mixture, a form of a dispersion in an aqueous solvent, and forms obtained byfurther drying or freezing said forms.
A dried mixture of lipids and a medicament can be produced by, for example,once dissolving lipid components and a medicament to be used in an organic solventsuch as chloroform and then subjecting the resulting solution to solidification underreduced pressure by using an evaporator or spray drying by using a spray dryer.Examples of a form in which a mixture of lipid membrane structures and amedicament are dispersed in an aqueous solvent include, but not particularly limitedthereto, multi-lamella liposomes, unilamella liposomes, O/W type emulsions, W/O/Wtype emulsions, spherical micelles, fibrous micelles, layered structures of irregularshapes and the like. A size of particles (particle diameter) as the mixture, acomposition of the aqueous solvent and the like are not particularly limited. Forexample, liposomes may have a size of 50 nm to 2 µm, spherical micelles may have asize of 5 to 100 nm, and emulsions may have a particle diameter of 50 nm to 5 µm. Aconcentration of the mixture in the aqueous solvent is also not particularly limited.Several methods are known as methods for producing a mixture of lipid membranestructures and a medicament in the form of dispersion in an aqueous solvent. It isnecessary to appropriately chose a suitable method depending on an available form ofthe mixture of lipid membrane structures and a medicament.<Production Method 1>
Production Method 1 is a method of adding an aqueous solvent to theaforementioned dried mixture of lipids and a medicament and emulsifying the mixtureby using an emulsifier such as homogenizer, ultrasonic emulsifier, high-pressureinjection emulsifier, or the like. When it is desired to control the size (particlediameter), extrusion (extrusion filtration) can be further performed under a highpressure by using a membrane filter having uniform pore sizes. In this method, inorder to prepare a dried mixture of lipids and a medicament first, it is necessary todissolve the medicament in an organic solvent, and the method has an advantage that it can make the best utilization of interactions between the medicament and lipidmembrane structures. Even when the lipid membrane structures have a layeredstructure, a medicament can enter into the inside of the multiple layers, and thus useof this method generally provides a higher retention ratio of the medicament in thelipid membrane structures.<Production Method 2>
Production Method 2 is a method of adding an aqueous solvent containing amedicament to dried lipid components obtained by dissolving the lipid components inan organic solvent and evaporating the organic solvent, and emulsifying the mixture.When it is desired to control the size (particle diameter), extrusion (extrusionfiltration) can be further performed under a high pressure by using a membrane filterhaving uniform pore sizes. This method can be used for a medicament that is hardlydissolved in an organic solvent, but can be dissolved in an aqueous solvent. When thelipid membrane structures are liposomes, they have an advantage that they can retaina medicament also in the part of internal aqueous phase.<Production Method 3>
Production Method 3 is a method of further adding an aqueous solventcontaining a medicament to lipid membrane structures such as liposomes, emulsions,micelles or layered structures already dispersed in an aqueous solvent. This methodis limitedly applied to a water-soluble medicament. The addition of a medicament toalready prepared lipid membrane structures is performed from the outside.Therefore, when the medicament is a polymer, the medicament cannot enter into theinside of the lipid membrane structures, and the medicament may be present in a formthat it binds to the surfaces of lipid membrane structures. When liposomes are usedas the lipid membrane structures, use of Production Method 3 may result in formationof a sandwich-like structure in which the medicament is sandwiched between liposomeparticles (generally called as a complex). An aqueous dispersion of lipid membranestructures alone is prepared beforehand in this production method. Therefore,decomposition of a medicament during the preparation need not be taken intoconsideration, and a control of the size (particle diameter) is also readily operated,which enables relatively easier preparation compared with Production Methods 1 and2. <Production Method 4>
Production Method 4 is a method of further adding an aqueous solventcontaining a medicament to a dried product obtained by once producing lipidmembrane structures dispersed in an aqueous solvent and then drying the same. Inthis method, a medicament is limited to a water-soluble medicament in the samemanner as Production Method 3. A significant difference from Production Method 3 isa mode of presence of the lipid membrane structures and a medicament. That is, inProduction Method 4, lipid membrane structures dispersed in an aqueous solvent areonce produced and further dried to obtain a dried product, and at this stage, the lipidmembrane structures are present in a state of a solid as fragments of lipid membranes.In order to allow the fragments of lipid membranes to be present in a solid state, it ispreferable to use an aqueous solution of a sugar, preferably an aqueous solution ofsucrose or aqueous solution of lactose, as the aqueous solvent as described above. Inthis method, when the aqueous solvent containing a medicament is added, hydration ofthe fragments of the lipid membranes present in a state of a solid quickly starts withthe invasion of water, and thus the lipid membrane structures can be reconstructed.At this time, a structure of a form in which a medicament is retained in the inside ofthe lipid membrane structures can be produced.
In Production Method 3, when a medicament is a polymer, the medicamentcannot enter into the inside of the lipid membrane structures, and is present in a modethat it binds to the surfaces of the lipid membrane structures. Production Method 4significantly differs in this point. In Production Method 4, an aqueous dispersion oflipid membrane structures alone is prepared beforehand, and therefore, decompositionof the medicament during the emulsification need not be taken into consideration, anda control of the size (particle diameter) is also easy attainable. For this reason, saidmethod enables relatively easier preparation compared with Production Methods 1 and2. Besides the above mentioned advantages, this method also has advantages thatstorage stability for a pharmaceutical preparation is easily secure, because the methoduses lyophilization or spray drying; when the dried preparation is rehydrated with anaqueous solution of a medicament, original size (particle diameter) can be reproduced;when a polymer medicament is used, the medicament can be easily retained in theinside of the lipid membrane structures and the like.
As other method for producing a mixture of lipid membrane structures and amedicament in a form of a dispersion in an aqueous solvent, a method well known asthat for producing liposomes, e.g., the reverse phase evaporation method or the like,may be separately used. When it is desired to control the size (particle diameter),extrusion (extrusion filtration) can be performed under a high pressure by using amembrane filter having uniform pore sizes. Further, examples of the method forfurther drying a dispersion, in which the aforementioned mixture of lipid membranestructures and a medicament is dispersed in an aqueous solvent, include lyophilizationand spray drying. As the aqueous solvent in this process, it is preferable to use anaqueous solution of a sugar, preferably an aqueous solution of sucrose or an aqueoussolution of lactose. Examples of the method for further freezing a dispersion, in whichthe aforementioned mixture of lipid membrane structures and a medicament isdispersed in an aqueous solvent, include ordinary freezing methods. As the aqueoussolvent in this process, it is preferable to use an aqueous solution of sugar or aqueoussolution of polyhydric alcohol in the same manner as the solution for the lipidmembrane structures alone.
Lipids that can be added to the pharmaceutical composition may be suitablychosen depending on a type of a medicament to be used and the like. The lipids areused in an amount of, for example, 0.1 to 1000 parts by mass, preferably 0.5 to 200parts by mass, based on 1 part by mass of a medicament when the medicament is not agene. When the medicament is a gene, the amount is preferably 1 to 500 nmol, morepreferably 10 to 200 nmol, with 1µg of a medicament (gene).
The method for use of the pharmaceutical composition of the present inventionwhich contains the lipid membrane structures may be suitably considered dependingon a form thereof. The administration route for humans is not particularly limited,and either oral administration or parenteral administration may be used. Examplesof dosage forms for oral administration include, for example, tablets, powders,granules, syrups, capsules, solutions for internal use and the like, and examples ofdosage forms for parenteral administration include, for example, injections, dripinfusion, eye drops, ointments, suppositories, suspensions, cataplasms, lotions,aerosols, plasters and the like. In the medicinal field, injections or drip infusion ispreferred among them, and as the administration method, intravenous injection, subcutaneous injection and intradermal injection, as well as local injection to targetedcells or organs are preferred. Further, as for the cosmetic field, examples of forms ofcosmetics include lotions, creams, toilet water, milky lotions, foams, foundations,lipsticks, packs, skin cleaning agents, shampoos, rinses, conditioners, hair tonics, hairliquids, hair creams and the like.Examples
The present invention will be explained more specifically with reference to thefollowing examples. However, the scope of the present invention is not limited tothese examples. In the chemical formulas shown in the following examples, theindications of PG(6), PG(8) and the like mean hexaglycerin, octaglycerin and the like,respectively, which are polyglycerin mixtures having average polymerization degreesof 6, 8 and the like, respectively.Synthesis Example 1(1) Preparation of distearoylphosphatidylethanolamine succinate
Distearoylphosphatidylethanolamine (20.0 g, 26.7 mmol) was added with 150mL of chloroform, stirred at 55°C, and added with 2.2 g (267 mmol) of sodium acetate toobtain a phospholipid solution in chloroform. The solution was added with 3.5 g (34.8mmol) of succinic anhydride and reacted at 55°C for 3 hours. Completion of thereaction was confirmed by thin layer chromatography (TLC) utilizing a silica gel platewhere no distearoylphosphatidylethanolamine was detected by ninhydrin coloration.As the developing solvent, a mixed solvent of chloroform and methanol at a volumeratio of 85:15 was used. After the reaction, the solution was filtered to remove sodiumacetate, and then the filtrate was concentrated. After the concentration of the filtrate, the residue was added with isopropyl alcohol (100 mL), and stirred at roomtemperature for 30 minutes. The crystals were collected by filtration, then washedwith hexane (80 mL), collected by filtration, and dried to obtain crystals ofdistearoylphosphatidylethanolamine succinate (20.5 g).Synthesis Example 2(2) Preparation of distearoylphosphatidylethanolamine glutarate
Distearoylphosphatidylethanolamine (20.0 g, 26.7 mmol) was added with 150mL of chloroform, stirred at 55°C, and added with 2.2 g (267 mmol) of sodium acetate toobtain a phospholipid solution in chloroform. The solution was added with 4.0 g (34.8mmol) of glutaric anhydride and reacted at 55°C for 3 hours. Completion of thereaction was confirmed by TLC in the same manner as described above. After thereaction, the solution was filtered to remove sodium acetate, and then the filtrate wasconcentrated. After the concentration of the filtrate, the residue was added withisopropyl alcohol (100 mL), and stirred at room temperature for 30 minutes. Thecrystals were collected by filtration, washed with hexane (80 mL), collected byfiltration, and dried to obtain crystals of distearoylphosphatidylethanolamineglutarate (19.8 g). Example 1(3) Preparation of hexaglycerol glutaryl distearoylphosphatidylethanolamine
Distearoylphosphatidylethanolamine glutarate (4.3 g, 5.0 mmol) was addedwith chloroform (25 mL) and stirred at 45°C. The chloroform solution was added with11.6 g (25 mmol) of hexaglycerin dissolved in dimethyl sulfoxide (10 mL), and thenadded with 2.1 g (10 mmol) of dicyclohexylcarbodiimide and 0.6 g (5.3 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by TLC, namely, confirmed by thin layerchromatography (TLC) utilizing a silica gel plate where nodistearoylphosphatidylethanolamine glutarate was detected. As the developingsolvent, a mixed solvent of chloroform, methanol and water at a volume ratio of 65:25:4was used. After the completion of the reaction, the deposited dicyclohexylurea wasremoved by filtration, and then the filtrate was passed through a cation exchange resin(DIAION SK1BH) filled in a column. The eluate was collected in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization. Theeluate was dehydrated over sodium sulfate, then filtered, and concentrated. Theresidue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 4.8 g of crystals of hexaglycerol glutaryldistearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂CH₂COO- derived from glutaric acid at δ 1.95,protons of methylene group of -NH(C=O)CH₂CH₂CH₂COO- at δ 2.29 and 2.31,methylene protons and methine protons derived from hexaglycerin at δ 3.2-4.5 wereobserved. Example 2(4) Preparation of octaglycerol glutaryl distearoylphosphatidylethanolamine
Distearoylphosphatidylethanolamine glutarate (4.3 g, 5.0 mmol) was addedwith chloroform (25 mL) and stirred at 45°C. This chloroform solution was added with15.3 g (25 mmol) of octaglycerin dissolved in dimethyl sulfoxide (20 mL), and thenadded with 2.1 g (10 mmol) of dicyclohexylcarbodiimide and 0.6 g (5.3 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by TLC in the same manner as describedabove. After the completion of the reaction, the deposited dicyclohexylurea wasremoved by filtration, and then the filtrate was passed through a cation exchange resin(DIAION SK1BH) filled in a column. The eluate was collected in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization. Theeluate was dehydrated over sodium sulfate, then filtered and concentrated. Theresidue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 4.5 g of crystals of octaglycerol glutaryldistearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂CH₂COO- derived from glutaric acid at δ 1.95,protons of methylene group of -NH(C=O)CH₂CH₂CH₂COO- at δ 2.29 and 2.31,methylene protons and methine protons derived from octaglycerin at δ 3.2-4.5 wereobserved. Example 3(5) Preparation of decaglycerol glutaryl distearoylphosphatidylethanolamine
Distearoylphosphatidylethanolamine glutarate (4.3 g, 5.0 mmol) was addedwith chloroform (25 mL) and stirred at 45°C. The chloroform solution was added with19.0 g (25 mmol) of decaglycerin dissolved in dimethyl sulfoxide (20 mL), and thenadded with 2.1 g (10 mmol) of dicyclohexylcarbodiimide and 0.6 g (5.3 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by TLC in the same manner as describedabove. After the completion of the reaction, the deposited dicyclohexylurea wasremoved by filtration, and then the filtrate was passed through a cation exchange resin(DIAION SK1BH) filled in a column. The eluate was collected in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization. Theeluate was dehydrated over sodium sulfate, then filtered and concentrated. Theresidue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 4.3 g of crystals of decaglycerol glutaryldistearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂CH₂COO- derived from glutaric acid at δ 1.95,protons of methylene group of -NH(C=O)CH₂CH₂CH₂COO- at δ 2.29 and 2.31,methylene protons and methine protons derived from decaglycerin at δ 3.2-4.5 wereobserved. Example 4(6) Preparation of octaglycerol succinyl distearoylphosphatidylethanolamine
Distearoylphosphatidylethanolamine succinate (4.2 g, 5.0 mmol) was addedwith chloroform (10 mL) and stirred at 45°C. The chloroform solution was added with15.3 g (25 mmol) of octaglycerin dissolved in dimethyl sulfoxide (20 mL), and thenadded with 2.1 g (10 mmol) of dicyclohexylcarbodiimide and 0.6 g (5.3 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by thin layer chromatography (TLC)utilizing a silica gel plate where no distearoylphosphatidylethanolamine succinate wasdetected. As the developing solvent, a mixed solvent of chloroform, methanol andwater at a volume ratio of 65:25:4 was used. After the completion of the reaction, thedeposited dicyclohexylurea was removed by filtration, and then the filtrate was passedthrough a cation exchange resin (DIAION SK1BH) filled in a column. The eluate wascollected in aqueous disodium hydrogenphosphate added with a small amount ofmethanol for neutralization. The eluate was dehydrated over sodium sulfate, thenfiltered and concentrated. The residue was crystallized 3 times fromchloroform/acetone/dimethyl sulfoxide, or acetone/dimethyl sulfoxide to obtain 4.8 g ofcrystals of octaglycerol succinyl distearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂COO- derived from succinic acid at δ 2.29 and2.31, methylene protons and methine protons derived from octaglycerin at δ 3.2-4.5were observed.Example 5(7) Preparation of tetradecaglycerol succinyl distearoylphosphatidylethanolamine
Distearoylphosphatidylethanolamine succinate (1.7 g, 2.0 mmol) was addedwith chloroform (10 mL) and stirred at 45°C. This chloroform solution was added with29.8 g (10 mmol) of tetradecaglycerin dissolved in dimethyl sulfoxide (40 mL), and thenadded with 0.8 g (4.0 mmol) of dicyclohexylcarbodiimide and 0.3 g (2.1 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by TLC in the same manner as describedabove. After the completion of the reaction, the deposited dicyclohexylurea wasremoved by filtration, and then the filtrate was passed through a cation exchange resin(DIAION SK1BH) filled in a column. The eluate was collected in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization. Theeluate was dehydrated over sodium sulfate, then filtered and concentrated. Theresidue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 3.8 g of crystals of tetradecaglycerol succinyldistearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂COO- derived from succinic acid at δ 2.29 and2.31, methylene protons and methine protons derived from tetradecaglycerin at δ3.2-4.5 were observed.Example 6: Evaluation as long circulating liposome in blood(1) Preparation of liposomes
Each of the lipids mentioned in each of the membrane compositions shown inTable 1 (Examples 1 to 5, Control Examples 1 to 4) were weighed in each ratio anddissolved in a chloroform/methanol mixture (2:1), then the organic solvents were evaporated by using an evaporator, and further the residue was dried under reducedpressure for 1 hour. Then, the dried lipids (lipid film) were added with 10 mL of 155mM aqueous ammonium sulfate (pH 5.5) heated at 65°C beforehand, and the mixturewas lightly stirred by using a vortex mixer on a hot water bath (until lipid wassubstantially peeled off from a recovery flask). This lipid dispersion was transferredto a homogenizer, homogenized for 10 strokes and sized by using polycarbonatemembrane filters with various pore sizes (0.2 µm x 3 times, 0.1 µm x 3 times, 0.05µm x 3 times and 0.03 µm x 3 times) to prepare a dispersion of empty liposomeshaving a particle diameter of about 100 nm.
In an amount of 4 mL of this empty liposome dispersion was diluted 2.5 timeswith physiological saline, and the resulting diluted liposome dispersion was placed inan ultracentrifugation tube and centrifuged at 65,000 rpm for 1 hour. Then, thesupernatant was discarded, and the precipitates were resuspended in physiologicalsaline to make the dispersion volume 10 mL, the volume of the liposome dispersionbefore the centrifugation (at this time point, the total lipid concentration was adjustedto 50 mM). The aforementioned empty liposome dispersion in which the externalaqueous phase was replaced with physiological saline (total lipid concentration: 50mM) and a doxorubicin solution (medicament concentration: 3.3 mg/mL physiologicalsaline) were heated beforehand at 60°C, and the empty liposome dispersion and thedoxorubicin solution were added at a volume ratio of 4:6 (i.e., final medicamentconcentration: 2.0 mg/mL, final lipid concentration, 20 mM) and incubated at 60°C for1 hour. The mixture was further cooled at room temperature to obtain adoxorubicin-containing liposome dispersion.(2) Physical properties of the liposome
The percentage of doxorubicin retained by the liposomes was obtained bycollecting a part of the aforementioned liposome dispersion, subjecting the sample togel filtration (Sephadex G-50, mobile phase was physiological saline), and thenquantifying doxorubicin in the liposome fraction eluted in the void volume by usingliquid chromatography. Further, particle diameter was determined by measurementbased on the quasi-elastic light scattering (QELS) method performed for a part of theaforementioned liposome dispersion. As a result, the percentage of doxorubicin, theactive ingredient retained by liposomes, was almost 100% in liposomes of Examples 2, 4 and 5, and Control Examples 1 and 2 as shown in Table 1. Therefore, each originalliposome dispersion was used without any treatment, and diluted 4/3 times withphysiological saline for the experiment utilizing rats described below (thus, finalmedicament concentration: 1.5 mg/mL, final lipid concentration: 15 mM). Further,the liposomes of Examples 1 and 3, and Control Examples 3 and 4 were subjected toultracentrifugation (65,000 rpm, 1 hour) to remove unencapsulated medicament in thesupernatant and then reconstituted with physiological saline so that a finalmedicament concentration of 1.5 mg/mL was obtained (thus, final lipid concentrationswere about 20.9 mM in Example 1, about 19.3 mM in Example 3, about 17.2 mM inControl Example 3, and about 18.7 mM in Control Example 4). The particlediameters of the liposomes were around 100 nm for all the examples.(3) Experiment for evaluation of circulating in blood in rats
An experiment for evaluation of circulating in blood was performed in SD malerats (6-week old) using Examples 1 to 5 and Control Examples 1 to 4 mentioned above.Each liposome dispersion was administered to rats from the cervical vein under etheranesthesia (each group consisted of 5 animals, dose: 7.5 mg doxorubicin/5 mL/kg), thenblood was collected in heparin (0.5 to 1 mL) from the cervical vein under etheranesthesia at each blood collection time (2, 4, 8, 24, 48, 72, 120, 168 hours) andsubjected to plasma skimming. Then, in a conventional manner, the blood waspretreated, and plasma medicament concentration was measured by HPLC. The AUC(0 to ∞) was calculated from the plasma medicament concentration obtained witheach formulation of liposome dispersion according to the trapezoidal rule. As shownin Table 1, AUCs larger by 1 order or more were obtained with the liposomeformulations containing the phospholipid derivatives of the present invention(Examples 1 to 5) compared with AUCs obtained with the liposomes of ControlExample 1 not containing the lipid derivative of the present invention, the liposomes ofControl Example 2 added only with the phospholipid portion (DSPE:distearoylphosphatidylethanolamine) of the lipid derivative of the present invention,and the liposomes of Control Examples 3 and 4 added with the polyglycerin lipidderivatives disclosed in Japanese Patent Unexamined Publication (KOKAI) No.6-22802 and literature (International Journal of Pharmacology, Vol. 111, page 103,1994), and thus clearly longer circulating in the blood was observed with the liposome formulations containing the phospholipid derivatives of the present invention. Liposome membrane composition Particle size (nm) Percentage of carried active ingredient (%) AUC_0~∞±S.D. (µg · hr/mL) Example 1 DSPE-PG(8)/HSPC/Cholesterol= 2.08 mM/11.28 mM/7.68 mM 92 71.8 3417±224 Example 2 DSPE-PG(40)/HSPC/Cholesterol= 0.72 mM/11.28 mM/7.68 mM 76 1000 3775±1038 (n=4) Example 3 DSPE-PG(6)Glu/HSPC/Cholesterol= 2.08 mM/11.28 mM/7.68 mM 94 77.6 4264±131 Example 4 DSPE-PG(8)Glu/HSPC/Cholesterol= 2.08 mM/11.28 mM/7.68 mM 78 96.6 4284±249 Example 5 DSPE-PG(10)Glu/HSPC/Cholesterol= 2.08 mM/11.28 mM/7.68 mM 83 100.0 4034±387 Control Example 1 HSPC/Cholesterol= 11.90 mM/8.10 mM 91 100.0 452±98 Control Example 2 DSPE/HSPC/Cholesterol= 1.04 mM/11.28 mM/7.68 mM mM/11.28 mM/7.68 mM 94 100.0 397±133 Control Example 3 DSPPG(4)/HSPC/Cholesterol= 1.04 mM/11.28 mM/7.68 mM 125 87.4 317±129 Control Example 4 DSPPG(6)/HSPC/Cholesterol= 1.04 mM/11.28 mM/7.68 mM 146 80.4 233±58 DSPE-PG(8): Synthesized in Example 4 DSPE-PG(40): Synthesized in Example 5 DSPE-PG(6)Glu: Synthesized in Example 1 DSPE-PG(8)Glu: Synthesized in Example 2 DSPE-PG(10)Glu: Synthesized in Example 3 HSPC: Hydrogenated soybean phosphatidylcholine DSPPG(4) and DSPPG(6): Polyglycerin lipid derivatives disclosed in Japanese Patent Unexamined Publication No. 6-228012 and literature (Int. J. Pharm., 111, 103 (1994)) Example 7: Preparation of skin toner (evaluation as solubilizer)
A skin toner was prepared by using octaglycerol glutaryl distearoylphosphatidylethanolamine of Synthesis Example 4. Specifically, among thebase materials in the composition shown in Table 2, glycerin and propylene glycol wereadded to purified water and uniformly dissolved. Other base materials were added toethanol, and the mixture was made uniform, then added to the aforementionedpurified water phase with stirring and solubilized to obtain a skin toner. Propylene glycol 5.0 wt% Glycerin 2.0 wt% Oleyl alcohol 0.5 wt% Hydrogenated soybean lecithin 0.5 wt% Ethanol 7.0 wt% Octaglycerol glutaryl distearoylphosphatidylethanolamine 2.0 wt% Tocopherol 0.02 wt% Perfume As required Preservative As required Purified water 73.0 wt% Example 8: Preparation of liposome emulsion (evaluation as dispersing agent forcosmetics)Method for preparing liposomes
In an amount of 645 mg of hydrogenated soybean phosphatidylcholine, 299 mgof cholesterol, 23 mg of myristic acid (molar ratio: 1:1:0.1) and octaglycerol glutaryldistearoylphosphatidylethanolamine were added so that the mixed lipid concentrationshould become 5% by mole, added with 10 to 11 mL of physiological saline heated at60°C beforehand so that the mixed lipid concentration was 10% by mass and stirred,and further mixed by using a homogenizer on a water bath at 60°C for 10 minutes toobtain a liposome solution. Among the base materials of the composition shown inTable 3, those of the oil phase containing an emulsifier were heated at 60°C anduniformly dissolved, and those of the aqueous phase using the liposome solution wereadded at the same temperature with stirring to obtain a liposome emulsion. Oil phase: Cetanol 2.0 wt% Vaseline 2.0 wt% Squalane 5.0 wt% Liquid paraffin 10.0 wt% Polyoxyethylene monooleic acid ester 2.0 wt% Tocopherol 0.02 wt% Perfume As required Preservative As required Aqueous phase: Propylene glycol 2.0 wt% Purified water 67.0 wt% Liposome solution 10.0 wt% Comparative Synthesis Example 1(1) Synthesis of monomethylpolyoxyethylenecarbamyl (molecular weight: 2000)distearoylphosphatidylethanolamine
Monomethoxypolyoxyethylene (molecular weight: 2000, 20 g, 10 mmol) wasadded with toluene (80 mL), and then refluxed by raising a temperature up to 110°C fordehydration. The reaction mixture was added with 1,1'-carbonyldiimidazole (1.95 g, 12mmol) and reacted at 40°C for 2 hours. The reaction mixture was added with pyridine(1.58 g, 20 mmol) and distearoylphosphatidylethanolamine (7 g, 9.36 mmol), and reactedat 65°C for 5 hours. The reaction mixture was added with hexane (300 mL) forcrystallization. The crystals were added with ethyl acetate (400 mL), dissolved at 65°C,stirred for 30 minutes, and then cooled to 5°C. The deposited crystals were collected byfiltration. This procedure using ethyl acetate was repeated again in a similar manner.The crystals were dissolved in ethyl acetate (400 mL), added with Kyoward #700 (1 g) asan adsorbent, and stirred at 65°C for 1 hour. The reaction mixture was filtered, andthen cooled to 5°C for crystallization. The crystals were washed with hexane (200 mL),collected by filtration, and dried to obtain 15.3 g (yield: 54.7%) of monomethylpolyoxyethylenecarbamyl distearoylphosphatidylethanolamine with a purityof 98.3%. The product was analyzed by thin layer chromatography (TLC) utilizing asilica gel plate. A mixed solvent of chloroform and methanol at a volume ratio of85:15 was used as a developing solvent, and substances contained were identified andquantified by coloration with iodine vapor on the basis of comparison with standardsubstances of known amounts.Example 9: Measurement of salt-tolerant effect (evaluation as surfactant)
Clouding point of a 1 mass % solution of tetradecaglycerol succinyldistearoylphosphatidylethanolamine obtained in Example 5, which was dissolved in 5mass % aqueous solution of sodium sulfate, was measured. As a result of themeasurement, clouding point could not be detected even when the temperature wasraised to 80°C.Comparative Example 1: Comparison of salt salt-tolerant effect (evaluation assurfactant)
Clouding point was measured for monomethylpolyoxyethylenecarbamyl(molecular weight: 2000) distearoylphosphatidylethanolamine obtained in ComparativeSynthesis Example 1 in the same manner as used in Example 9. As a result of themeasurement, clouding point was found to be 50.0°C. Thus, it was revealed that thephospholipid derivative of the present invention exhibited high salt tolerance.Example 10 (evaluation as surfactant)Preparation of polymer micelle solution of hydrogenated soybean phosphatidylcholineusing octaglycerol glutaryl distearoylphosphatidylethanolamine
Distilled water (5 mL) was added with hydrogenated soybeanphosphatidylcholine (0.1 g, 0.13 mmol) and octaglycerol glutaryldistearoylphosphatidylethanolamine (1 g, 0.17 mmol), and mixed by stirring. Theresulting uniform mixed solution was gradually added with distilled water (95 mL) withstirring to obtain a transparent uniform polymer micelle solution. Particle sizedistribution in the obtained solution was measured by using a particle sizer (NICOMPModel 370, produced by Nozaki & Co., Ltd.). As a result, mean particle size was found to be 40 nm. The resulting polymer micelle solution was left for one month at roomtemperature. After 3 months, the polymer micelle solution had a condition of a uniformpolymer micelle solution and gave no change under visual inspection and no precipitates.Example 11Synthesis of octaglycerol nonaglutarate (compound of the following formula wherein k =8, k2 = 9, and k3 = 1)
Octaglycerin (6.1 g, 0.01 mol) was dispersed in dimethyl sulfoxide (50 mL),added with 9.0 g (0.11 mol) of sodium acetate, warmed to 70°C, and then added with 11.4g (0.1 mol) of glutaric anhydride and reacted for 12 hours. After completion of thereaction, sodium acetate was removed by filtration, and dimethyl sulfoxide wasevaporated under reduced pressure by using an evaporator to obtain 15.9 g ofoctaglycerol nonaglutarate.
Acid value and hydroxyl value of the resulting compound were measured. Theacid value was found to be 310.8, and hydroxyl value was 36.1. On the basis of theseresults, it was revealed that about 9 hydroxyl groups of octaglycerin were glutarated, andabout one hydroxyl group existed. Thus the compound obtained was proved to beoctaglycerol nonaglutarate.
By ¹H-NMR (CDCl₃), protons of methyl group of -O(C=O)CH₂CH₂CH ₂COO-derivedfrom glutaric acid at δ 1.97, protons of methylene group of-O(C=O)CH ₂CH₂CH ₂COO- at δ 2.41 and 2.44, methylene protons and methine protonsderived from octaglycerin at δ 3.2-4.6 were observed.Synthesis of octaglycerol heptaglutaryl phosphatidylethanolamine glutarate (compoundof the following formula wherein k = 8, k1 = 1, k2 = 8, and k3 = 1)
Distearoylphosphatidylethanolamine (9.4.g, 0.012 mmol) was added withchloroform (150 mL) and stirred at 45°C. This phospholipid/chloroform solution wasadded with 15.9 g (0.097 mol) of the aforementioned crude octaglycerol glutaratedissolved in dimethyl sulfoxide (15 mL), and then added with 2.4 g (0.012 mol) ofdicyclohexylcarbodiimide, 1.3 g (0.012 mol) of triethylamine and 1.4 g (0.012 mol) ofN-hydroxysuccinimide, and reacted for 3 hours.
Completion of the reaction was confirmed by TLC, specifically completion wasconfirmed by thin layer chromatography (TLC) utilizing a silica gel plate where nodistearoylphosphatidylethanolamine was detected. As the developing solvent, amixed solvent of chloroform, methanol and water at a volume ratio of 65:25:4 was used.After the completion of the reaction, the deposited dicyclohexylurea was removed byfiltration, and then the filtrate was passed through a cation exchange resin (DIAIONSK1BH) filled in a column. The eluate was received in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization. Theeluate was dehydrated over sodium sulfate, then filtered, and concentrated. Theresidue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 18.1 g of octaglycerol glutaryldistearoylphosphatidylethanolamine.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH ₂CH₂COO- derived from glutaric acid at δ 1.95,protons of methylene group of -NH(C=O)CH ₂CH₂CH ₂COO- at δ 2.29 and 2.31,methylene protons and methine protons derived from octaglycerin at δ 3.2-4.5 were observed.Example 12(8) Preparation of hexaglycerol distearoylphosphatidylethanolamine succinate ester
Distearoylphosphatidylethanolamine succinate (4.2 g, 5.0 mmol) was addedwith chloroform (10 mL) and stirred at 45°C. The chloroform solution was added with11.6 g (25 mmol) of hexaglycerin dissolved in dimethyl sulfoxide (20 mL), and thenadded with 2.1 g (1.0 mmol) of dicyclohexylcarbodiimide and 0.64 g (5.3 mmol) ofdimethylaminopyridine. The reaction was performed at 45°C for 2 hours.Completion of the reaction was confirmed by TLC in the same manner as describedabove.
After the completion of the reaction, the deposited dicyclohexylurea wasremoved by filtration, and then the filtrate was passed through a cation exchange resin(DIAION SK1BH) filled in a column. The eluate was received in aqueous disodiumhydrogenphosphate added with a small amount of methanol for neutralization.
The eluate was dehydrated over sodium sulfate, then filtered and concentrated.The residue was crystallized 3 times from chloroform/acetone/dimethyl sulfoxide, oracetone/dimethyl sulfoxide to obtain 4.7 g of crystals of hexaglyceroldistearoylphosphatidylethanolamine succinate ester.
By ¹H-NMR (CDCl₃), protons of methyl group at the end of the stearoyl groupat δ 0.88, protons of methylene group of the stearoyl group at δ 1.26, protons ofmethylene group of -NH(C=O)CH₂CH₂COO- derived from succinic acid at δ 2.29 and2.31, methylene protons and methine protons derived from hexaglycerin at δ 3.2-4.5were observed.(Evaluation as solubilizer)
Cyclosporin A (25 mg, produced by Sigma) was weighed in a sample tube, anddissolved in dimethyl sulfoxide (1 mL) to prepare a cyclosporin A/dimethyl sulfoxidesolution. The octaglycerol succinyl distearoylphosphatidylethanolamine (30 mg)obtained in Example 4 was added with 200 µ L of the cyclosporin A/dimethyl sulfoxidesolution obtained above, and completely dissolved by warming. The resulting solutionwas added with 800 µL of purified water, and sufficiently stirred.
In the same manner, experiment was also performed with the hexaglyceroldistearoylphosphatidylethanolamine succinate ester obtained in Example 12.
Then, experiment was also performed similarly with medroxyprogesteroneacetate (produced by Sigma).
Medroxyprogesterone acetate (2.5 mg) was weighed in a sample tube, anddissolved in DMSO (1 mL) to prepare a cyclosporin A/DMSO solution. The octaglycerolsuccinyl distearoylphosphatidylethanolamine (30 mg) obtained in Example 4 was addedwith 200 µL of the cyclosporin A/DMSO solution obtained above, and completelydissolved by warming. The solution obtained was added with 800 µL of purified water,and sufficiently stirred.
In the same manner, experiment was also performed with the hexaglyceroldistearoylphosphatidylethanolamine succinate ester obtained in Example 12.
Complete solubilization was observed by visual inspection, and the results wereindicated with ○ when complete dissolution was obtained, or with × when anyinsolubility was observed.○ : Transparent× : Turbid
For Control Examples 14 and 15, the polyglycerin lipid derivatives disclosed inJapanese Patent Unexamined Publication (KOKAI) No. 6-22802 and the literature(International Journal of Pharmacology, Vol. 111, page 103, 1994) were used.
For Control Example 16, Cremophor EL (polyoxyl 35 castor oil, produced bySigma) was used.
All the results are shown in Table 4. Cyclosporin A Medroxyprogesterone acetate Example 13 DSPE-PG(6) ○ ○ Example 14 DSPE-PG(8) ○ ○ Control Example 14 DSPPG(6) × × Control Example 15 DSPPG(8) × × Control Example 16 Cremophor EL × × DSPE-PG(6): Synthesized in Example 12 DSPE-PG(8): Synthesized in Example 4 DSPPG(4) and DSPPG(6): Polyglycerin lipid derivatives disclosed in Japanese Patent Unexamined Publication No. 6-228012 and the literature (Int. J. Pharm., 111, 103 (1994)) Industrial Applicability
The phospholipid derivative of the present invention is highly safe for livingbodies and useful as a surfactant, solubilizer, or dispersing agent in the fields ofcosmetics and the like. When the phospholipid derivative of the present invention,which is a polyglycerin derivative, is used for preparing a lipid membrane structuresuch as liposome, aggregation of microparticles in an aqueous medium is preventedwithout causing instability of the lipid membrane structure, and a stable solution statecan be obtained. Further, a liposome containing the phospholipid derivative of thepresent invention is characterized to have a longer circulating time in blood.

权利要求:
Claims (19)
[1] A phospholipid derivative represented by the following formula (1):
[2] The phospholipid derivative according to claim 1, wherein k1 satisfies 1≦k1 ≦2.
[3] The phospholipid derivative according to claim 1 or 2, wherein k2 satisfies 0≦ k2 ≦ 1.
[4] The phospholipid derivative according to any one of claims 1 to 3, whereink1, k2, and k3 satisfy 8 ≦ k1 + k2 + k3 ≦ 52.
[5] The phospholipid derivative according to any one of claims 1 to 4, whereinR¹CO and R²CO independently represent an acyl group having 12 to 20 carbon atoms.
[6] The phospholipid derivative according to any one of claims 1 to 5, whereink2 is 0.
[7] The phospholipid derivative according to claim 6, wherein a and b represent0.
[8] The phospholipid derivative according to any one of claims 1 to 5, wherein k2 satisfies 0 < k2.
[9] A lipid membrane structure comprising the phospholipid derivativeaccording to any one of claims 1 to 8.
[10] The lipid membrane structure according to claim 9, which is a liposome.
[11] A surfactant comprising the phospholipid derivative according to any oneof claims 1 to 8.
[12] A solubilizer comprising the phospholipid derivative according to any oneof claims 1 to 8.
[13] A dispersing agent comprising the phospholipid derivative according to anyone of claims 1 to 8.
[14] A method for producing the phospholipid derivative according to claim 1,which comprises the step of reacting a compound represented by the following formula(2):
[15] A method for producing the phospholipid derivative according to claim 1,which comprises the following steps:
(A) the step of reacting a polyglycerin with a dibasic acid or a halogenated carboxylic acid to obtain a carboxylated polyglycerin; and
(B) the step of reacting the carboxylated polyglycerin obtained in the step (A) with aphospholipid.
[16] A method for producing the phospholipid derivative according to claim 1,which comprises the following steps:
(A) the step of reacting a polyglycerin with a halogenated carboxylic acid ester andhydrolyzing the resulting ester compound to obtain a carboxylated polyglycerin; and
(B) the step of reacting the carboxylated polyglycerin obtained in the step (A) with aphospholipid.
[17] A method for producing the phospholipid derivative according to any one ofclaims 1 to 7, which comprises the step of reacting a polyglycerin derivativerepresented by the following formula (4):
[18] A pharmaceutical composition containing the lipid membrane structureaccording to claim 9 retaining a medicament.
[19] The pharmaceutical composition according to claim 18, wherein themedicament is an antitumor agent.

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JP4403303B2|2010-01-27|

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EP1591447B1|2014-11-26|

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CA2513144A1|2004-07-22|

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CA2513144C|2012-03-13|

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AU2003289070A1|2004-07-29|

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US7524981B2|2009-04-28|

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KR101007828B1|2011-01-13|

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WO2004060899A1|2004-07-22|

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JPWO2004060899A1|2006-05-11|

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优先权:

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申请号 | 申请日 | 专利标题

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JP2003000330||2003-01-06||

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JP2003000330||2003-01-06||

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PCT/JP2003/015969|WO2004060899A1|2003-01-06|2003-12-12|Phospholipid derivatives and process for the production there|

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