JP2010235450A - Lipid derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new lipid derivative that can be used in an amount similar to that of an ordinary phospholipid PEG, can expand a hydrated layer on a surface of a lipid vesicle, can improve stability of the lipid vesicle and can be readily produced. <P>SOLUTION: The lipid derivative includes a residue of a compound chosen from glucamine, N-methylglucamine, gluconic acid and glucoheptonic acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an amphiphilic compound having three or more alkyl chains serving as hydrophobic groups and one or two polyethylene glycol chains as hydrophilic groups.

  The amphiphilic compound of the present invention is useful for stably fixing a water-soluble polymer, a polysaccharide, and a water-soluble protein to a lipid vesicle, a cell surface, or a hydrophobic surface. Furthermore, specific recognition ability for cells and proteins can be imparted by modifying lipid vesicles and microspheres.

  Lipid vesicles are expected as carriers in the field of drug delivery systems (DDS) from the viewpoint of safety such as high biocompatibility, and various studies have been made so far. However, lipid vesicles are also known to have many problems that they are easily destabilized by aggregation or fusion, or have low blood retention. Therefore, mixing lipids and cholesterol with constituent lipids of the endoplasmic reticulum has been performed to suppress aggregation and fusion and stabilize the lipid endoplasmic reticulum (Patent Document 1). Furthermore, the solution has been achieved by modifying the surface of the endoplasmic reticulum with polyethylene glycol or carbohydrate. Surfaces modified with water-soluble polymer chains such as polyethylene glycol are highly biocompatible and can be used as materials for artificial organs, artificial cells, and artificial blood. Furthermore, lipid vesicles having a hydrated layer formed by a water-soluble polymer such as polyethylene glycol are less likely to be taken into the reticuloendothelial system (RES) in the blood, and have a blood retention property. I know it ’s good. When the retention in the blood is good, circulating in the blood increases the probability of accumulation in damaged cells such as cancer cells, and the DDS function can be improved. In addition, by introducing a functional molecule into polyethylene glycol, a characteristic function can be added to the lipid vesicle, and targeting to the affected area can be expected. Therefore, polyethylene glycol derivatives are expected as useful compounds in the DDS field.

  When applied to lipid vesicles, polyethylene glycol derivatives are useful as a material for DDS because they are effective not only in suppressing aggregation and the like, but also in improving blood retention. Has been done. As a polyethylene glycol derivative imparted to a lipid vesicle, a lipid polyethylene glycol (lipid PEG) combined with a hydrophobic compound is widely used. Examples thereof include diacylphosphatidylethanolamine PEG (phospholipid PEG) and Examples include cholesterol PEG. Lipid PEG incorporates a hydrophobic portion into the lipid bilayer membrane, and projects polyethylene glycol, which is a water-soluble portion, onto the lipid surface to coat the surface. In view of this, studies have been made to increase the amount of lipid PEG added and increase the molecular weight of the polyethylene glycol chain in order to improve the blood retention and targeting ability. However, it has been reported that when the addition amount or the molecular weight is excessively increased, the balance between hydrophilicity and hydrophobicity is lost, the packing of the phospholipid membrane is weakened, and the lipid PEG is detached from the lipid vesicles (non-patent). Reference 1). In addition, it has been reported that the stability of lipid vesicles deteriorates due to this elimination (Non-patent Document 2).

  On the other hand, in Patent Document 2, in order to stably fix to the surface to the lipid endoplasmic reticulum, a large number of hydrophobic groups are introduced into the branch side end of the dendritic structure (dendron) which is a dendrimer constituent unit, and the core portion There is a description of an amphiphilic compound having a dendritic branched structure in which a hydrophilic group is introduced into the only substituent in the above. However, for the synthesis of this compound with a large number of hydrophobic groups, the first step is to purify when the hydrophobic group is introduced at the branch end of the dendritic structure, and the second is to increase the branching. Purification is also required when a group is introduced, and thirdly, purification is necessary even after introducing a hydrophilic group into the only substituent in the core part. . Moreover, the evaluation to the endoplasmic reticulum surface as this compound is not made | formed. Therefore, it is an object of the present invention to provide a lipid derivative that can be easily produced, increases the hydration layer on the surface of the lipid vesicle, and increases the stability of the lipid vesicle.

Japanese Patent Laid-Open No. 7-145038 Japanese Patent No. 3181276

J. et al. R. Silvius and M.M. J. et al. Zuckermann, Biochemistry, 32, 3153, 1993 Biophysical Journal, 74, 1371, 1998

  The problem to be solved by the present invention is that it can be used in the same amount as that of ordinary phospholipid PEG, and can increase the hydration layer on the surface of the lipid vesicle, thereby stabilizing the stability of the lipid vesicle. It is an object of the present invention to provide a novel lipid derivative that can be improved and can be easily produced.

  More specifically, it is an object to be solved to provide a lipid derivative that can be suitably used not only in the field of DDS such as lipid vesicles but also in solubilization and dispersion of physiologically active substances.

  As a result of intensive studies to solve the above problems, the present inventors have succeeded in providing a novel lipid derivative.

  The lipid derivative of the present invention has 3 to 6 hydrophobic groups incorporated into the bilayer membrane of the lipid vesicle, and after being incorporated into the bilayer membrane due to its hydrophobic interaction, it is stably present in the membrane. be able to. In addition, since hydrophobic interaction is strong, it can exist stably once incorporated into the membrane. Therefore, even if the chain length of polyethylene glycol is increased in order to increase the hydration layer, the hydrophobic group portion is stably incorporated into the membrane, so that it does not fall out.

Therefore, the present invention is as follows.
[1] The following general formula (I)

A lipid derivative represented by:
(In the formula, Z is a residue of a compound having a hydroxyl group having 4 to 8 carbon atoms, or a residue of a compound having 4 to 8 carbon atoms having one or two amino groups or carboxyl groups together with the hydroxyl group. , R represents an alkyl group or alkenyl group having 4 to 24 carbon atoms, m is 0 to 6, a is 0 or 1, POLY is a linear or branched polyoxyalkylene, POLY has a molecular weight of 200 to 100,000, k ₁ is 1 or 2, k ₂ is 3 to 6, k ₃ is 0 to ₃ , 4 ≦ k ₁ + k ₂ + k ₃ ≦ 8, and X is , O, NHC (═O) O, NHC (═O), N (CH ₃ ) C (═O) O, N (CH ₃ ) C (═O), C (═O) NH .)
[2] The lipid derivative according to [1], wherein Z is a residue of a compound selected from glucamine, N-methylglucamine, gluconic acid, and glucoheptonic acid.
[3] The lipid derivative according to the above [1] or [2], wherein POLY is methoxypolyoxyethylene.
[4] The lipid derivative according to [1] or [2], wherein POLY is a linear polyoxyethylene having a functional group selected from a maleimide group, an aldehyde group, and an N-succinimide ester group at the terminal.
[5] The lipid derivative according to the above [1] or [2], wherein POLY is branched polyoxyethylene.
[6] The lipid derivative according to any one of [1] to [5], wherein a = 1.
[7] k ₃ is zero, the lipid derivative according to any one of [1] to [6].
[8] A lipid vesicle comprising the lipid derivative according to any one of [1] to [7].
[9] The following general formula (II)

The polyoxyalkylene derivative represented by these.
(Wherein m and POLY are the same as above, Z ′ is a residue of a compound having 4 to 8 carbon atoms having one or two of an amino group and a carboxyl group together with a hydroxyl group, and X ′ is NHC. (═O) O, NHC (═O), N (CH ₃ ) C (═O) O, N (CH ₃ ) C (═O), C (═O) NH, a group selected from k, ₄ is 1 or 2, _{k 5} is 3-6).

  In the lipid derivative of the present invention represented by the general formula (I), Z is a residue of a compound having a hydroxyl group having 4 to 8 carbon atoms, or 1 or 2 of either an amino group or a carboxyl group together with the hydroxyl group. It is a residue of a compound having 4 to 8 carbon atoms.

  Examples of the compound having a hydroxyl group having 4 to 8 carbon atoms include erythritol, pentaerythritol, pentitol, hexitol, heptitol, octitol, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, mannitol, dipentaerythritol and the like. Is mentioned. Preferably, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, mannitol, pentitol, heptitol, more preferably triglycerin, pentaglycerin, pentitol, heptitol, even more preferably pentitol, Heptitol. These may be natural products or synthetic products and have various isomers, and xylitol is particularly preferable among pentitols. Moreover, you may use individually or in mixture of 2 or more types.

  On the other hand, as a compound of 4 to 8 carbon atoms having one or two of an amino group or a carboxyl group together with a hydroxyl group, mucoic acid (carboxyl group: 2, hydroxyl group: 4), gluconic acid (carboxyl group: 1, hydroxyl group) : 5), glucamine (amino group: 1, hydroxyl group: 5), N-methylglucamine (amino group: 1, hydroxyl group: 5), glucoheptonic acid (carboxyl group: 1, hydroxyl group: 6) and the like. It is not limited to these. Preferred are gluconic acid, glucamine and glucoheptonic acid, and more preferred is glucamine.

  A compound having 4 to 8 carbon atoms having either one or two amino groups or carboxyl groups only needs to have one or two amino groups or carboxyl groups in the structure. After protecting 3 to 6 hydroxyl groups in a compound having 4 to 8 hydroxyl groups by a known method and derivatizing the remaining 1 or 2 hydroxyl groups into a carboxyl group or an amino group by a known method It can be obtained by a deprotection method or the like.

  R is an alkyl group or alkenyl group having 4 to 24 carbon atoms, preferably an alkyl group or alkenyl group having 11 to 23 carbon atoms. The alkyl group and alkenyl group may be linear or branched, and the alkenyl group may have 1 to 3 double bonds.

  This R is usually derived from a fatty acid or an aliphatic alcohol. a represents 0 or 1, and when a = 1, specific examples of the acyl group that is RCO include butyric acid, isobutyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid Examples include acyl groups derived from saturated and unsaturated linear or branched fatty acids such as acids, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, arachidic acid, behenic acid, erucic acid, and lignoceric acid. it can.

  When a = 0, specific examples of R include n-propanol, butanol, pentanol, hexanol, heptanol, octanol, 2-ethylhexanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, penta Examples include decanol, hexadecanol, heptadecanol, octadecanol, nonadecanol, icosanol, heicosanol, docosanol, tricosanol, derived alkyl group or alkenyl group, which may be linear or branched It may be.

X is a bond derived from an amino group or a carboxyl group of a compound having a hydroxyl group of 4 to 8 carbon atoms, or a compound of 4 to 8 carbon atoms having one or two of an amino group or a carboxyl group together with the hydroxyl group, O, -NHC (= O) - ( amide _{bond), - N (CH 3)} C (= O) - ( amide bond), - C (= O) NH- ( amide bond), - NHC (= O) O- (urethane _{bond), - N (CH 3)} C (= O) O- ( urethane bond), - a O- (ether bond).

  POLY represents polyoxyalkylene, and examples thereof include polyoxyethylene, polyoxypropylene, polyoxytrimethylene, polyoxybutylene and the like. Among these, polyoxyethylene and polyoxypropylene are preferable, and a polyoxyethylene group is particularly preferable. One oxyalkylene group may be used alone, or two or more types may be combined, and there is no limitation on the combination. Further, it may be a block shape or a random shape.

  POLY is polyoxyalkylene which may be linear or branched.

  The structure of POLY is more specifically shown below, but is not limited thereto.

  For example, the linear polyoxyalkylene is a polyoxyethylene derivative shown below.

  Here, n is the number of added moles of polyoxyalkylene when the molecular weight of polyoxyalkylene is 200 to 100,000.

  Examples of the branched polyoxyalkylene are polyoxyethylene derivatives shown below.

  The terminal of the polyoxyalkylene may be a reactive group capable of binding a drug, an antibody or the like, or may be an inactive functional group to which a drug, an antibody or the like cannot bind.

  For example, reactive groups include maleimide group, acetal group, aldehyde group, thiol group, protected thiol group, succinic acid succinimide ester group, glutaric acid succinimide ester group, amino group, protected amino group Oxyamino group, p-nitrophenyl carbonate group, hydroxyl group and the like. Preferred are a methoxy group, a maleimide group, an acetal group, an aldehyde group, a glutaric acid succinimide ester group, and an amino group, and more preferred are a methoxy group, a maleimide group, an acetal group, an aldehyde group, and a glutaric acid succinimide ester. It is a group.

  Examples of the inactive functional group include a methoxy group, an ethoxy group, and a t-butoxy group, and a methoxy group is preferable.

  In addition, a polyoxyalkylene represented by POLY and a reactive group or an inert group may have a linking group containing an alkylene group such as a methylene group, an ethylene group or a propylene group, an amide group or an ester group. Good.

  The molecular weight of POLY is 200-100000, preferably 500-40000, more preferably 1000-10000, and still more preferably 1000-5000. When the molecular weight of the polyoxyalkylene is less than 200, when the polyoxyalkylene-lipid derivative of the present invention is used as an additive to the lipid vesicle, the effect of stabilization is small because the hydrophobicity is too strong. On the other hand, when it is more than 100,000, the viscosity of the polyoxyalkylene-lipid derivative is increased during production, and workability is lowered, which is not preferable.

  m is an integer of 0 to 6, preferably 0 to 3. When m is 0, it indicates that POLY and X are directly bonded.

k ₁ , k ₂ , and k ₃ are each an integer, k ₁ is 1 or 2, k ₂ is 3 to 6, k ₃ is 0 to 3, and the sum of k ₁ , k ₂ , and k ₃ is 3 ≦ k ₁ + k ₂ + k ₃ ≦ 6.

  The production method of the compound of the present invention represented by the general formula (I) is not particularly limited, but an activated derivative of a polyethylene glycol compound, for example, a polyethylene glycol carbonate derivative, a polyethylene glycol carboxylic acid derivative, a polyethylene glycol amine derivative and the like, After obtaining a compound represented by the general formula (II) by reacting with a compound having 4 to 8 carbon atoms having either one or two amino groups or carboxyl groups together with a hydroxyl group, fatty acid, fatty acid chloride, or fatty acid Can be prepared by reacting with an anhydride.

In general formula (II), m and POLY are the same as described above. Z ′ is a residue of a compound having 4 to 8 carbon atoms having one or two amino groups or carboxyl groups together with a hydroxyl group, and X ′ is NHC (═O) O, NHC (═O), N ( A group selected from CH ₃ ) C (═O) O, N (CH ₃ ) C (═O), and C (═O) NH. k ₄ and k ₅ are each an integer, k ₄ is 1 or 2, k ₅ is 3 to 6, and the sum of k ₄ and k ₅ is 4 ≦ k ₄ + k ₅ ≦ 8.

  The polyoxyalkylene-lipid derivative of the present invention can be contained as a constituent component of a lipid vesicle formed of phospholipid or the like. The lipid membrane structure contains sterols such as phospholipids, cholesterol and cholestanol, other fatty acids having saturated and unsaturated acyl groups having 8 to 22 carbon atoms, and antioxidants such as α-tocopherol. Also good. Phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2- Dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen and the like can be mentioned, and these can be used alone or in combination of two or more. The fatty acid residues of these phospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms. Specifically, lauric acid, myristic acid, palmitic acid, Mention may be made of acyl groups derived from fatty acids such as stearic acid, oleic acid and linoleic acid. Moreover, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.

  The production method of the compound of the present invention represented by the general formula (I) is not particularly limited, but an activated derivative of a polyethylene glycol compound, for example, a polyethylene glycol carbonate derivative, a polyethylene glycol carboxylic acid derivative, a polyethylene glycol amine derivative and the like, It can be produced by reacting a compound having 4 to 8 carbon atoms having either one or two amino groups or carboxyl groups and then lipidating the remaining hydroxyl group.

  In addition, as a method for producing the present compound, at least one of compounds having a hydroxyl group having 4 to 8 carbon atoms as Z is protected with a protecting group, and after, for example, ethylene oxide is polymerized to the remaining hydroxyl group, It can also be produced by a method in which a hydroxyl group is converted into a desired functional group and then deprotected, and the resulting hydroxyl group is lipidated.

  In addition, lipid vesicles using the lipid derivatives of the present invention can encapsulate drugs and physiologically active substances, react with functional groups of lipid derivatives, and bind to various proteins and antibodies. Genes can also be encapsulated. These are not particularly limited in the type of medicine and the type of gene. The amount of the lipid derivative of the present invention to be incorporated into the lipid vesicles may be an amount sufficient for drug efficacy and gene expression.

  As the medicament, for example, a compound used as an antitumor agent is preferable, and examples thereof include irinotecan hydrochloride, paclitaxel, doxorubicin hydrochloride, mitomycin C, vincristine sulfate and the like.

  Examples of the gene include oligonucleotides, DNA, RNA, and the like, for example, genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes, cytokines, and the like.

  The form of the lipid vesicle containing the lipid derivative of the present invention is not particularly limited. For example, a form in which the lipid mixture is dissolved in an organic solvent such as chloroform and the solvent is removed by drying using an evaporator, or the lipid mixture is used as an aqueous solvent. Examples thereof include a dispersed form and a form obtained by freeze-drying vesicles dispersed in an aqueous solvent.

  Examples of the form in which the lipid mixture is dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, and string micelles. Liposomes are preferred. When an aqueous solvent is used, simple water may be used, or a mixed solvent containing a small amount of alcohol or the like may be used. Preferably, pharmacopoeia water for injection, distilled water and the like are used. At this time, a physiologically active substance, protein, buffer substance, various salts, plasma and the like may be dissolved in an aqueous solvent to form a solution.

  The form in which the lipid mixture is dispersed in an aqueous solvent is not particularly limited, but the dispersion solution can be emulsified using a homogenizer, a high-pressure jet emulsifier, or the like, or obtained as a liposome having a uniform particle size by an extruder, a liposome, or the like. I can do it.

  The mixed dried product of lipids and medicine is, for example, the lipid components and medicine to be used are once dissolved in an organic solvent such as chloroform, and then this is dried under reduced pressure using an evaporator or spray dried using a spray dryer. Can be manufactured.

  A production method in which a mixture of a lipid mixture and a medicine is dispersed in an aqueous solvent can be carried out by a known method and can be appropriately selected. Although it is not limited to these as a manufacturing method, For example, the method of adding a mixture of a lipid mixture and a medicine to an aqueous solvent, and emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, etc. A method of adding an aqueous solvent containing a medicine to emulsify, a method of adding an aqueous solvent containing a medicine to a lipid vesicle such as a liposome, emulsion, or micelle dispersed in an aqueous solvent, a method of adding a lipid vesicle dispersed in an aqueous solvent Examples include a method of adding an aqueous solvent containing a medicine to a dried product.

  When it is desired to control the size (particle diameter) of the lipid vesicles, extrusion may be performed using an extruder or the like under a high pressure using a membrane filter having a uniform pore diameter.

  Examples of the present invention will be described below.

Example 1
Synthesis of methylpolyoxyethyleneoxycarbonyl glucamine (PEG molecular weight: 2000)

  D-glucamine (8.15 g, 45 mmol) and dimethyl sulfoxide (50 g) were placed in a 300 mL flask and dissolved at 40 ° C. Methyl polyoxyethylene p-nitrophenyl carbonate (SUNBRIGHT MENP-20H: molecular weight 2000, 50 g, 25 mmol) was dissolved in dimethyl sulfoxide (100 g) at 40 ° C., and then added to the above solution. After the reaction for 3 hours, the solution was sampled, diluted, and then subjected to TLC (thin layer chromatography: chloroform / methanol = 85/15) to confirm that the raw material monomethyl polyoxyethylene p-nitrophenyl carbonate had disappeared. Toluene (317 g) was added to the reaction solution, methyl t-butyl ether (hereinafter referred to as MTBE) (616 g) was added, and the mixture was cooled to 5 ° C. After crystal filtration, ethyl acetate (800 g) was added, and the temperature was raised at 45 ° C., followed by cooling to room temperature. Insoluble matters were filtered with a glass filter (GF-75), and the crystals were filtered after cooling to 5 ° C. Further, after the above purification was repeated, the crystals were washed with hexane (800 g), filtered and dried to obtain 50 g of the target compound. The obtained compound was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15), and confirmed by iodine color development, it was 1 spot.

^{As a} characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 3.38 (s, C H ₃ O—, 3H), 3.4-3.9 (m, — (C H ₂ C H ₂ O) ) n -), 4.22 (m , -NH (C = O) OC H 2 -) was confirmed.

Example 2
Synthesis of methylpolyoxyethyleneoxycarbamyl glucamine pentastearate (PEG molecular weight: 2000)

  Stearic acid (10.7 g, 37.6 mmol) and toluene (100 g) were placed in a 300 mL flask and dissolved at 40 ° C. Dicyclohexylcarbodiimide (3.87 g, 18.8 mmol) was added, and methylpolyoxyethyleneoxycarbonyl glucamine (5.0 g, 2.5 mmol) obtained in Example 1 above, dimethylaminopyridine (1.0 g, 8.1 mmol) were obtained. ) And allowed to react for 3 hours. After filtering the reaction solution, the solvent was distilled off with an evaporator. The residue was dissolved in acetone (40 mL), hexane (200 mL) was added, and the mixture was cooled to 5 ° C. After crystal filtration, acetone (100 mL) and hexane (200 mL) were added and cooled to 5 ° C. After crystal filtration, the same procedure was repeated once more. After crystal filtration, the crystals were washed with hexane (200 mL), filtered and dried to obtain 5.3 g of the target compound.

  Moreover, the analysis of the residual fatty acid was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15) and analyzed by color development of copper phosphate sulfate. As a result, the residual stearic acid was 0.05% or less.

Moreover, as a characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 0.88 (t, C H ₃ (CH ₂ ) _16- , 15 H), 3.38 (s, C H ₃ O—, 3 H _{), 3.4-3.9 (m, - (} C H 2 C H 2 O) n-) was confirmed.

Example 3
Synthesis of methylpolyoxyethyleneoxycarbonyl glucamine (PEG molecular weight: 5000)

  D-glucamine (2.90 g, 16 mmol) and dimethyl sulfoxide (50 g) were placed in a 300 mL flask and dissolved at 40 ° C. Methyl polyoxyethylene p-nitrophenyl carbonate (molecular weight 5000, 50 g, 10 mmol) was dissolved in dimethyl sulfoxide (130 g) at 40 ° C. and then added to the above solution. After the reaction for 3 hours, the solution was sampled, diluted, and then subjected to TLC (thin layer chromatography: chloroform / methanol = 85/15) to confirm that the raw material monomethyl polyoxyethylene p-nitrophenyl carbonate had disappeared. Toluene (285 g) was added to the reaction solution, MTBE (553 g) was added, and the mixture was cooled to 5 ° C. After crystal filtration, ethyl acetate (800 g) was added, and the temperature was raised at 45 ° C., followed by cooling to room temperature. Insoluble matters were filtered with a glass filter (GF-75), and the crystals were filtered after cooling to 5 ° C. Further, after the above purification was repeated, the crystals were washed with hexane (800 g), filtered and dried to obtain 46.5 g of the target compound.

  The obtained compound was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15), and confirmed by iodine color development, it was 1 spot.

^{As a} characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 3.38 (s, C H ₃ O—, 3H), 3.4-3.9 (m, — (C H ₂ C H ₂ O) ) n -), 4.22 (m , -NH (C = O) OC H 2 -) was confirmed.

Example 4
Synthesis of methylpolyoxyethyleneoxycarbonyl glucamine pentastearate (PEG molecular weight: 5000)

  Stearic acid (12.8 g, 45 mmol) and toluene (300 g) were placed in a 300 mL flask and dissolved at 40 ° C. Dicyclohexylcarbodiimide (4.70 g, 22.5 mmol) was added, and methylpolyoxyethyleneoxycarbonyl glucamine (15.0 g, 3 mmol) and dimethylaminopyridine (1.2 g, 9.8 mmol) obtained in Example 3 above were added. The mixture was allowed to react for 2 hours. After filtering the reaction solution, the solvent was distilled off with an evaporator. The residue was dissolved in acetone (120 mL), hexane (600 mL) was added, and the mixture was cooled to 5 ° C. After crystal filtration, acetone (300 mL) and hexane (600 mL) were added and cooled to 5 ° C. After crystal filtration, the same procedure was repeated once more. After crystal filtration, the crystals were washed with hexane (600 mL), filtered and dried to obtain 12.7 g of the target compound.

  The analysis of the residual fatty acid was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15) and analyzed by color development of copper phosphate sulfate. As a result, the residual stearic acid was 0.05% or less.

Moreover, as a characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 0.88 (t, C H ₃ (CH ₂ ) _16- , 15 H), 3.38 (s, C H ₃ O—, 3 H _{), 3.4-3.9 (m, - (} C H 2 C H 2 O) n-) was confirmed.

Example 5
Synthesis of 3- (3′-maleimido-1′-oxopropyl) aminopropyl-polyoxyethyleneoxycarbonyl glucamine (PEG molecular weight: 5000)

  D-glucamine (2.90 g, 16 mmol) and dimethyl sulfoxide (50 g) were placed in a 300 mL flask and dissolved at 40 ° C. SUNBRIGHT MA-050TS (NOF Corporation) (molecular weight 5000, 50 g, 10 mmol) was dissolved in dimethyl sulfoxide (130 g) at 40 ° C. and then added to the above solution. After the reaction for 3 hours, the solution was sampled, diluted, and then subjected to TLC (thin layer chromatography: chloroform / methanol = 85/15) to confirm that the raw material monomethyl polyoxyethylene p-nitrophenyl carbonate had disappeared. Toluene (285 g) was added to the reaction solution, MTBE (553 g) was added, and the mixture was cooled to 5 ° C. After crystal filtration, ethyl acetate (800 g) was added, and the temperature was raised at 45 ° C., followed by cooling to room temperature. Insoluble matters were filtered with a glass filter (GF-75), and the crystals were filtered after cooling to 5 ° C. Further, after the above purification was repeated, the crystals were washed with hexane (800 g), filtered and dried to obtain 45.1 g of the target compound.

  The obtained compound was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15), and confirmed by iodine color development, it was 1 spot.

The peaks of features than _{^{1 H-NMR (CDCl 3)}} , 3.4-3.9 (m, - (C H 2 C H 2 O) n -), 4.22 (m, -NH (C _{= O) OC H 2 -)} , 6.71 (s, -C H = C H -, 2H), were confirmed.

Example 6
Synthesis of 3- (3′-maleimido-1′-oxopropyl) aminopropyl-polyoxyethyleneoxycarbonyl glucamine pentastearate (PEG molecular weight: 5000)

  Stearic acid (12.8 g, 45 mmol) and toluene (300 g) were placed in a 300 mL flask and dissolved at 40 ° C. Dicyclohexylcarbodiimide (4.70 g, 22.5 mmol) was added and 3- (maleimido-1-oxopropyl) aminopropyl-polyoxyethyleneoxycarbonyl glucamine (15.0 g, 3 mmol), dimethyl obtained in Example 5 above was added. Aminopyridine (1.2 g, 9.8 mmol) was added and allowed to react for 2 hours. After filtering the reaction solution, the solvent was distilled off with an evaporator. The residue was dissolved in acetone (120 mL), hexane (600 mL) was added, and the mixture was cooled to 5 ° C. After crystal filtration, acetone (300 mL) and hexane (600 mL) were added and cooled to 5 ° C. After crystal filtration, the same procedure was repeated once more. After crystal filtration, the crystals were washed with hexane (600 mL), filtered and dried to obtain 12.7 g of the target compound.

  The analysis of the residual fatty acid was developed by TLC (thin layer chromatography: chloroform / methanol = 85/15) and analyzed by color development of copper phosphate sulfate. As a result, the residual stearic acid was 0.05% or less.

Moreover, as a characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 0.88 (t, C H ₃ (CH ₂ ) ₁₆ —, 15 H), 1.75 (quint, —CH ₂ C H ₂ CH _{2 NH-, 2H), 3.4-3.9 (} m, - (C H 2 C H 2 O) n -), 6.71 (s, -C H = C H -, 2H) confirmed the .

Comparative Example 1
Synthesis of monomethyl polyoxyethylene p-nitrophenyl carbonate

  SUNBRIGHT MEH-10H (molecular weight 1000: 100 g, 0.1 mol) and toluene (110 g) were added, dehydrated at 110 ° C., and water-containing toluene (33 g) was distilled off. After cooling to 60 ° C., triethylamine (12.1 g, 0.12 mol) was added, p-nitrophenyl chloroformate (22.2 g, 0.11 mmol) was added, and the mixture was reacted for 4 hours. After cooling to 40 ° C., it was diluted with toluene (120 g), and triethylamine hydrochloride was filtered. Ethyl acetate (200 g) and hexane (300 g) were added to the filtrate and cooled to 5 ° C. After crystal filtration, after dissolving in ethyl acetate (200 g), hexane (300 g) was added and cooled to 5 ° C. After crystal filtration, the crystals were washed with hexane (300 g). After crystal filtration, the product was dried under reduced pressure to obtain monomethyl polyoxyethylene p-nitrophenyl carbonate (96 g) as the target product.

^{As a} characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 3.38 (s, C H ₃ O—, 3H), 3.4-3.9 (m, — (C H ₂ C H ₂ O) _{) n -), 4.43 (-OCH} 2 C H 2 OC (= O) O-, 2H), 7.40 (d, -C H = CH-, 2H), 8.28 (d, -CH = C H- , 2H).

Comparative Example 2
Synthesis of monomethyl polyoxyethylene carbamyl distearoylphosphatidylethanolamine

  Monomethyl polyoxyethylene p-nitrophenyl carbonate (30 g, 0.1 mol) and toluene (150 g) obtained in Comparative Example 1 were added, and the temperature was raised to 68 ° C. Distearoylphosphatidylethanolamine (26.9 g, 0.12 mol) and sodium carbonate (63.9 g, 0.20 mol) were added and reacted for 5 hours. After cooling to 30 ° C., sodium carbonate was filtered. The filtrate was concentrated and dried with an evaporator. After dissolving in ethyl acetate (30 g), methyl t-butyl ether (270 g) was added and cooled to 5 ° C. After crystal filtration, crystallization of ethyl acetate (30 g) / methyl t-butyl ether (270 g) was repeated twice. After crystal filtration, the crystals were washed with hexane (300 g). After crystal filtration, the product was dried under reduced pressure to obtain monomethyl polyoxyethylene carbamyl distearoylphosphatidylethanolamine (45 g), which was the target product.

^{As a} characteristic peak from ¹ H-NMR (CDCl ₃ ), δ 0.88 (s, C H ₃ (CH ₂ ) ₁₆ CO _{2 —} , 6H), 1.25 (m, CH ₃ (C H ₂ ) _{14 , 56H), 3.38 (s,} C H 3 O-, 3H), 3.4-3.9 (m, - (C H 2 C H 2 O) n -), 5.21 (m, - _{OC (= O) C H (} CH 2 -) 2, 1H) was confirmed.

(Evaluation as a liposome)
[1] Preparation of Liposome Solution After dissolving 6.059 g (7.7 mmol) of hydrogenated soybean phosphatidylcholine and 1.0 g (2.6 mmol) of cholesterol in 200 mL of chloroform, the solvent was distilled off by an evaporator, and vacuum was further applied for 6 hours. Dried. 0.07 g of this dried lipid was weighed, added with 20 mL of water to be hydrated, and lightly stirred with a vortex mixer while attached to a hot water bath. This lipid dispersion was sized using a polycarbonate membrane filter having a pore size of 0.2 μm to prepare a liposome solution (0.2 μm × 2 times). Five sets of the liposome liquids were prepared, and the liposome liquids (i) to (v) were prepared.

Example A
[2] Preparation of polyoxyethylene-modified liposome solution Separately, the compound represented by the general formula (2) obtained in Example 2 was methylpolyoxyethyleneoxycarbonyl glucamine pentastearate 0.086 g (2.57 × 10 ⁻² mmol) was dissolved in 5 mL of water to prepare a dispersion (5.1 mM). This dispersion was added to the liposome liquid (i) and stirred at 60 ° C. for 1 hour to prepare polyoxyethylene-modified liposome (A). (Lipid concentration 4 mM, PEG concentration 1 mM)

Example B
Methyl polyoxyethyleneoxycarbonyl glucamine pentastearate 0.163 g (2.57 × 10 ⁻² mmol), which is the compound represented by the general formula (4) obtained in Example 4, was dissolved in 5 mL of water. A dispersion (5.1 mM) was prepared. This dispersion was added to the liposome liquid (ii) and stirred at 60 ° C. for 1 hour to prepare polyoxyethylene-modified liposome (B). (Lipid concentration 4 mM, PEG concentration 1 mM)

Comparative Examples C, D, E
A compound represented by the general formula (8) of Comparative Example 2, a compound represented by the following general formula (9) (polyethylene glycol molecular weight 2000), a compound represented by the following general formula (10) (polyethylene glycol molecular weight 615). Were used in an amount of 2.57 × 10 ⁻² mmol (0.046 g, 0.072 g, 0.026 g, respectively) and dissolved in 5 mL of water, respectively, to prepare three types of dispersions (5.1 mM). 5 mL of these dispersions were added to each of the liposome liquids (iii) to (v), and stirred at 60 ° C. for 1 hour to prepare three types of polyoxyethylene-modified liposomes (C, D, E). (Lipid concentration 4 mM, PEG concentration 1 mM)

(Study of stability of polyoxyethylene modified liposome)
In order to evaluate the stability of polyoxyethylene-modified liposomes, the five polyoxyethylene-modified liposomes obtained in Examples A and B and Comparative Examples C, D, and E were subjected to a low-temperature incubator (LTI-700E: 40 ° C.). EYELA), and the stability at 1, 1.5, 2, 3 months was evaluated. The results are shown in the table.

  As a result of the stability evaluation of the polyoxyethylene-modified liposomes at 40 ° C., the polyoxyethylene-modified liposomes (A) and (B) of Example A and Example B containing the lipid derivative of the present invention remained after 3 months. It was stable.

  On the other hand, polyoxyethylene-modified liposomes (C) and (D) using conventional lipid derivatives were aggregated in 1.5 months and 2 months, respectively. The polyoxyethylene modified liposome (E) was solubilized in one month.

  From the above, it has been clarified that the polyoxyethylene-modified liposome containing the lipid derivative of the present invention is more stable than the polyoxyethylene-modified liposome using the conventional lipid derivative.

Claims (9)

The following general formula (I)

A lipid derivative represented by:
(In the formula, Z is a residue of a compound having a hydroxyl group having 4 to 8 carbon atoms, or a residue of a compound having 4 to 8 carbon atoms having one or two amino groups or carboxyl groups together with the hydroxyl group. , R represents an alkyl group or alkenyl group having 4 to 24 carbon atoms, m is 0 to 6, a is 0 or 1, POLY is a linear or branched polyoxyalkylene, POLY has a molecular weight of 200 to 100,000, k ₁ is 1 or 2, k ₂ is 3 to 6, k ₃ is 0 to ₃ , 4 ≦ k ₁ + k ₂ + k ₃ ≦ 8, and X is , O, NHC (═O) O, NHC (═O), N (CH ₃ ) C (═O) O, N (CH ₃ ) C (═O), C (═O) NH .)   The lipid derivative according to claim 1, wherein Z is a residue of a compound selected from glucamine, N-methylglucamine, gluconic acid, and glucoheptonic acid.   The lipid derivative according to claim 1 or 2, wherein POLY is methoxypolyoxyethylene.   The lipid derivative according to claim 1 or 2, wherein POLY is a linear polyoxyethylene having a functional group selected from a maleimide group, an aldehyde group, and an N-succinimide ester group at the terminal.   The lipid derivative according to claim 1 or 2, wherein POLY is branched polyoxyethylene.   The lipid derivative according to claim 1, wherein a = 1. k ₃ is zero, the lipid derivative of any one of claims 1 to 6.   A lipid vesicle comprising the lipid derivative according to any one of claims 1 to 7. The following general formula (II)

The polyoxyalkylene derivative represented by these.
(Wherein m and POLY are the same as above, Z ′ is a residue of a compound having 4 to 8 carbon atoms having one or two of an amino group and a carboxyl group together with a hydroxyl group, and X ′ is NHC. (═O) O, NHC (═O), N (CH ₃ ) C (═O) O, N (CH ₃ ) C (═O), C (═O) NH, a group selected from k, ₄ is 1 or 2, _{k 5} is 3-6).