JP2002363278A - Polyalkylene oxide-modified phospholipid and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polyalkylene oxide-modified phospholipid having a small amount of impurities such as a base having nitrogen atom and a monoacylphospholipid. SOLUTION: The polyalkylene oxide-modified phospholipid containing <=3 wt.% monoacylphospholipids and <=0.02 wt.% bases containing nitrogen atoms is expressed by formula (1) wherein, R<1> CO, R<2> CO are each independently a 4-24C acyl; (k) is 1-6; R<3> O is a 2-4C oxyalkylene; (n) is 10-800 averaged molar number of addition of the 2-4C oxyalkylene; M is H, sodium or potassium; X is a 1-3C divalent hydrocarbon or C(C=O)(CH2 )q [wherein, (q) is 1-4]; and (p) is 0 or 1 and when (p) is 0, Y is H or a 1-4C alkyl, and when (p) is 1, Y is H, amino, carboxyl, aldehyde, glycidyl or thiol}.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel and useful polyalkylene oxide-modified phospholipid, a method for producing the same, and a use thereof. The present invention relates to an alkylene oxide-modified phospholipid, its production method and use.

[0002]

2. Description of the Related Art In recent years, liposome preparations such as anticancer agents have been widely studied, and liposomes have been extensively modified with water-soluble polymers in order to improve their retention in blood. As one of them, a liposome-modified polyethylene oxide-modified phospholipid is used. Since these are used for pharmaceutical purposes, it is preferable that they have as little impurities as possible or no impurities.

[0003] In the synthesis reaction of liposome-modified polyethylene oxide-modified phospholipids, a nitrogen-containing base such as triethylamine is often used as a catalyst. In this case, it was necessary to acidify the system in order to remove the base present in excess after the reaction. During this step, the liposome-modified polyethylene oxide-modified phospholipids deteriorated, and it was difficult to obtain a high-purity product. A nitrogen-containing base such as triethylamine often has an ammonia odor or a unique odor.
It is desirable not to use such a base in the working environment.

A method for synthesizing polyethylene oxide-modified phospholipids is described in MCWoodle (Biochimica et Biophysica A
cta, 1105, 193-200 (1992) and S. Zalipsky (Bioconjuga
te Chem, 4, 296-299 (1994)). Specifically, in an organic solvent, the terminal of polyethylene glycol is activated using 1,1′-carbonyldiimidazole or disuccinimidyl carbonate, and then reacted with a phospholipid in the presence of a base such as triethylamine. This is a method of obtaining polyethylene oxide-modified phospholipids by purifying by reverse phase silica gel chromatography, dialysis, or the like. Also in this method, the system is made acidic immediately after the reaction in order to remove excess base such as triethylamine. At this time, monoacyl phospholipids (generally referred to as lysophospholipids) are produced. This is highly biotoxic and poses a problem when used in pharmaceutical applications, for example, as a drug delivery system. In addition, if reverse phase silica gel chromatography or dialysis is performed in the purification step after synthesis, the product may degrade during elution from the column (such as the production of monoacyl phospholipids described above), or the product may undergo hydrolysis during dialysis. Occurs. Therefore, it is difficult to obtain a high-purity product, which is a problem from an industrial viewpoint.

As described above, in the conventional method, it was difficult to obtain a high-purity product because a base such as triethylamine or the like was contained, or lysophospholipid was contained when the base was removed. Further, in the conventional method, it is difficult to develop the product into industrial production due to the low product yield and the large amount of solvent used. for that reason,
A simple method of synthesizing a highly pure polyalkylene oxide-modified phospholipid that does not contain impurities such as a base having a nitrogen atom such as triethylamine or a monoacyl phospholipid is desired.

[0006] Since phospholipids have an excellent effect as both an emulsifier and a humectant, their use in cosmetics as well as pharmaceuticals has been studied a lot. In the cosmetics field, its use has been studied as liposomes and by the addition of other surfactants. However, application to emulsions, lotions, and the like has been difficult due to the problem of solubility, which has the problem of not dissolving when the added amount is increased. Therefore, the amount of the surfactant other than the surfactant of the present invention must be increased.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a polyalkylene oxide-modified phospholipid having a low impurity content, such as a base having a nitrogen atom or a monoacyl phospholipid, and a high purity, a method for producing the same, and a use thereof. That is. Further, the polyalkylene oxide-modified phospholipid of the present invention has a phospholipid effect and is soluble in an aqueous solution, so that it can be used as a surfactant.

[0008]

The present invention relates to the following polyalkylene oxide-modified phospholipids, a method for producing the same, and uses. (1) The content of the monoacyl phospholipid is 3% by weight or less, and the content of the base having a nitrogen atom is 0.02% or less.
A polyalkylene oxide-modified phospholipid represented by the following formula (1), which is not more than% by weight

Embedded image (In the formula (1), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, k is 1 to 6, R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is a carbon atom. The average number of moles of the oxyalkylene group represented by the formulas 2 to 4 is 10 to 800. M is a hydrogen atom, sodium or potassium, X
Is a divalent hydrocarbon group having 1 to 3 carbon atoms or -C (= O)
(CH ₂ ) q- (where q is 1 to 4), and p is 0 or 1. When p is 0, Y is a hydrogen atom or a group having 1 to 4 carbon atoms.
Is an alkyl group. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. (2) The polyalkylene oxide-modified phospholipid according to (1), wherein the content of the monoacyl phospholipid is 2% by weight or less. (3) The polyalkylene oxide-modified phospholipid according to the above (1), wherein p is 0, Y is a methyl group, and the content of the monoacylphospholipid is 0.5% by weight or less. (4) A method for producing a polyalkylene oxide-modified phospholipid having the following step (A). Step (A): A solution containing an activated form of a polyalkylene oxide compound represented by the following formula (2) and a phospholipid represented by the following formula (3), wherein the aqueous solution is an alkali metal salt showing alkalinity and containing no nitrogen. Reacting in an organic solvent in the presence of a salt.

Embedded image (In the formula (2), R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, n is an average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and is a number of 10 to 800. X is a carbon number. 1-3
Divalent hydrocarbon group or _{-C (= O) (CH 2} ) q- of (here q 1 to 4), p is 0 or 1. When p is 0, Y is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. Z is an activity imparting group. )

Embedded image (In the formula (3), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, and k is 1 to 6.) (5) R ¹ CO and R ² CO each have 12 carbon atoms. The production method according to the above (4), which is an acyl group of from 20 to 20. (6) The production method according to the above (4) or (5), wherein p is 0 and Y is a methyl group. (7) The production method according to any one of the above (4) to (6), wherein the solid salt used in the step (A) is sodium carbonate, and the organic solvent is toluene or chloroform. (8) The method according to any one of the above (4) to (7), comprising the following step (B) after the step (A). Step (B): a step of removing unreacted phospholipid represented by the formula (3) using ethyl acetate or acetone. (9) The method according to any one of the above (4) to (8), comprising the following step (C) after the step (A). Step (C): a step of performing recrystallization using ethyl acetate and / or acetone as a solvent. (10) The method according to (9), wherein in step (C), at least one compound selected from the group consisting of aliphatic hydrocarbons having 5 to 8 carbon atoms and ether is used as a solvent. (11) The method according to (9) or (10), wherein the step (C) is performed after the step (B). (12) The method according to any one of the above (4) to (11), comprising the following step (D) after the step (A). Step (D): a step of performing purification using an adsorbent. (13) The method according to the above (12), wherein the adsorbent used in the step (D) is an alkaline earth metal oxide, an alkaline earth metal hydroxide, an adsorbent containing aluminum or silicon, or activated carbon. . (14) The method according to the above (12) or (13), wherein the step (D) is performed after the step (B). (15) The polyalkylene oxide-modified phospholipid is represented by the following formula (1) in which the content of a monoacylphospholipid is 3% by weight or less and the content of a base having a nitrogen atom is 0.02% by weight or less. (4) which is a compound to be
-The production method according to any one of (14) to (14).

Embedded image (In the formula (1), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, k is 1 to 6, R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is a carbon atom. The average number of moles of the oxyalkylene group represented by the formulas 2 to 4 is 10 to 800. M is a hydrogen atom, sodium or potassium, X
Is a divalent hydrocarbon group having 1 to 3 carbon atoms or -C (= O)
(CH ₂ ) q- (where q is 1 to 4), and p is 0 or 1. When p is 0, Y is a hydrogen atom or carbon number 1-4.
Is an alkyl group. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. (16) A surfactant comprising the polyalkylene oxide-modified phospholipid according to any one of (1) to (3). (17) A liposome-forming agent comprising the polyalkylene oxide-modified phospholipid according to any one of (1) to (3). (18) A liposome comprising the polyalkylene oxide-modified phospholipid according to any one of (1) to (3). (19) An amphipathic chemical modifier for a physiologically active substance comprising the polyalkylene oxide-modified phospholipid according to any one of the above (1) to (3). (20) An emulsifier for a physiologically active substance comprising the polyalkylene oxide-modified phospholipid according to any one of (1) to (3). (21) A solubilizing agent for a physiologically active substance comprising the polyalkylene oxide-modified phospholipid according to any one of (1) to (3). (22) A polymer micelle containing the polyalkylene oxide-modified phospholipid according to any one of (1) to (3).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the polyalkylene oxide-modified phospholipid of the present invention represented by the above formula (1), R ¹ C
O and R ² CO have 4 to 24 carbon atoms, preferably 12 to 24 carbon atoms.
20 acyl groups. This acyl group is usually derived from a fatty acid. Specific examples of R ¹ CO and R ² CO include, for example, butyric acid, isobutyric acid, caproic acid, caprylic acid,
Capric, lauric, myristic, palmitic, palmitoleic, stearic, isostearic, oleic, linoleic, arachiic, behenic,
Acyl groups derived from saturated and unsaturated linear or branched fatty acids such as erucic acid and lignoceric acid can be mentioned. R ¹ CO and R ² CO may be the same or different from each other.

The oxyalkylene group represented by R ³ O in the above formula (1) is an oxyalkylene group having 2 to 4, preferably 2 or 3 carbon atoms. Examples of R ³ O include an oxyethylene group, an oxypropylene group, an oxytrimethylene group, and an oxybutylene group. Among these, an oxyethylene group and an oxypropylene group are preferred, and an oxyethylene group is particularly preferred. There are n oxyalkylene groups in the molecule as many as n. These oxyalkylene groups may be used alone or in a combination of two or more, and the combination is not limited. It may be in a block shape or a random shape.

In the above formula (1), n represents the average number of moles of the added oxyalkylene group, and is 10 to 800, preferably 20 to 500. When n is 10 or more, the delivery effect is enhanced when the polyalkylene oxide-modified phospholipid of the present invention is used in a drug delivery system. When n is 800 or less, the viscosity of the polyalkylene oxide compound represented by the formula (2), which is a raw material, does not increase so much when producing a polyalkylene oxide-modified phospholipid, so that workability is improved.

In the above formula (1), M is a hydrogen atom, sodium or potassium. The polyalkylene oxide-modified phospholipid of the present invention represented by the above formula (1) may be a mixture thereof. Sodium or potassium which forms a salt with a phosphate group is usually contained in a physiological saline buffer used for preparing a drug or the like, and is also present in a living body. Therefore, even if the polyalkylene oxide-modified phospholipid of the present invention is used in a drug delivery system, there are few problems such as toxicity in the body. M is a metal ion other than sodium or potassium,
For example, a divalent metal ion such as calcium or magnesium is not preferable because it has a structure in which two phospholipid molecules are associated.

In the above formula (1), k is an integer of 1 to 6, preferably 1 to 4, more preferably 2 to 4, and still more preferably 2. Wherein X in formula (1) is a divalent hydrocarbon radical or a -C 1 to 3 carbon atoms (= O) (CH ₂₎
q- (where q is 1 to 4). -CH ₂ Concrete examples of the hydrocarbon group is _{-, - CH 2 CH 2 -} , - (CH 2)
_{3 -, - CH (CH 3} ) CH 2 - and the like. p is 0
Or 1.

When p is 0 in the formula (1), Y is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably an alkyl group. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group.
Of these, a methyl group is preferred. When p is 1,
Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group, and is preferably an amino group. Xp-Y is preferably a methyl group, an ethyl group, a propyl group, and an aminomethyl group, and particularly preferably a methyl group.

The polyalkylene oxide-modified phospholipid of the present invention has a low content of impurities such as a monoacylphospholipid and a base having a nitrogen atom, which is a residue derived from a catalyst. The content of the monoacyl phospholipid in the polyalkylene oxide-modified phospholipid of the present invention is 3% by weight or less, preferably 2% by weight or less, more preferably 0.5% by weight or less.

The content of the base having a nitrogen atom is 0.02% by weight or less, more preferably 0.1% by weight.
The content is not more than 01% by weight, more preferably substantially not contained. The base having a nitrogen atom is a residue derived from the catalyst, for example, a base derived from a base such as triethylamine and has at least one nitrogen atom. Examples of the base having a nitrogen atom include pyridine, triethylamine, diisopropylamine, dimethylaminopyridine, imidazole, diethylamine, diisobutylamine, tri-n-octylamine, di-2-ethylhexylamine and the like. Among these,
In particular, it is desired that triethylamine is low in toxicity. Although the phospholipid represented by the formula (3) has a nitrogen atom, it is not a base having a nitrogen atom because it is neutralized in the molecule.

The polyalkylene oxide-modified phospholipid of the present invention has a low content of monoacyl phospholipid, which is considered to be highly biotoxic, and a low content of impurities having a nitrogen atom such as triethylamine. . Further, the polyalkylene oxide-modified phospholipid of the present invention contains a monovalent metal cation of sodium ion or potassium ion which is also present in the living body, and therefore has high safety for the living body. As described above, since it is highly safe for living bodies, it can be used as a surfactant, particularly a living body surfactant. Among them, it can be suitably used as a surfactant for a physiologically active substance.

The surfactant of the present invention is a surfactant containing the polyalkylene oxide-modified phospholipid of the present invention. In general, it is desirable to use 0.01 to 30% by weight of a polyalkylene oxide-modified phospholipid with respect to the entire composition such as a composition containing a surfactant, and preferably 0.1 to 30% by weight.
It is desirable to use 1 to 5% by weight. In addition,
The amount of the surfactant used is appropriately selected depending on the purpose of use of the surfactant, the difference in the composition, and the like.

As the surfactant of the present invention, one or more polyalkylene oxide-modified phospholipids represented by the above formula (1) may be used in combination, or may be used in combination with another surfactant. . The surfactant of the present invention comprises 1) an emulsifier for emulsifying a water-insoluble or hardly soluble target substance in a dispersion medium such as water or a buffer, and 2) a water-insoluble or hardly soluble target substance in water.
A solubilizing agent that dissolves in a dispersion medium such as a buffer, 3) a dispersant that disperses an insoluble or hardly soluble target substance in water or a dispersion medium such as a buffer, 4) a hydrophilic group and a hydrophobic group It can be suitably used as an amphiphilic chemical modifier for introducing a group.

Emulsions, solubilizations or dispersions can be obtained using the surfactants of the present invention. When the surfactant of the present invention is used as an emulsifier, solubilizer or dispersant, the emulsifier, solubilizer or dispersant may use only the surfactant of the present invention, and may be used for emulsification, solubilization or dispersion. It may contain other known components used.

The form of the solubilizing solution or dispersion is not limited, and a physiologically active substance or the like is dissolved in a dispersing solution in which a physiologically active substance or the like is dissolved in a dispersion medium such as water or a buffer, or a dispersion medium such as water or a buffer. And the like. The form of the emulsion or the solubilizing solution is not limited, and may be a micelle solution formed by the surfactant of the present invention, that is, a micelle solution containing a lipophilic physiologically active substance inside the micelle, or water or a buffer solution. Emulsion solutions in which the dispersion particles of the surfactant of the present invention and a lipophilic physiologically active substance or the like are present as colloidal particles or larger particles in the dispersion medium are exemplified. Examples of the micelle solution include those having a dispersed particle diameter of 10 to 300 nm, particularly as a polymer micelle solution. The emulsion solution is an oil-in-water (O / W) type in which a physiologically active substance is blended in an oil phase or a multilayer oil-in-water (W / O / W) in which a physiologically active substance is blended in an aqueous phase.
It may be a type.

Since the surfactant of the present invention has hydrophilicity derived from a polyoxyalkylene group and hydrophobicity derived from an acyl group, it can be used as an emulsifier or a solubilizing agent, and can be stored stably. Also excellent in nature. Furthermore, it is composed of a phospholipid equivalent to the constituents of a living cell and a polyoxyalkylene group having low toxicity, and has a low content of a monoacylphospholipid which is said to be biotoxic, so that the safety is higher. By using an emulsifier or a solubilizer containing such a polyalkylene oxide-modified phospholipid, for example, a highly safe emulsion or solubilization solution of a physiologically active substance can be obtained.

The substance to be emulsified, solubilized or dispersed includes physiologically active substances, fat-soluble substances and the like. Specific examples of the physiologically active substance include enzymes, antibodies, other proteins, sugars, lipids, glycoproteins, glycolipids, and hormones. Examples of the fat-soluble substance include lipids such as phospholipids, fat-soluble drugs such as taxol, and higher alcohols, ester oils, triglycerin, tocopherol, and higher fatty acids which are usually used in an oil phase. The surfactant of the present invention may contain other known components such as polyhydric alcohols such as glycerin and propylene glycol, fatty acid esters, preservatives, and antioxidants, as long as the object of the present invention is not impaired. it can.

Using the polymer surfactant containing a polyoxyalkylene chain of the present invention, the particle size is 10 to 300 nm.
Can be obtained as a polymer micelle solution in which the fine particles are present as micelles. As the polymer micelle, the above formula (1)
May be used alone, or may contain other known components. As described later, a polymer surfactant in which another known polymer is bonded via the reactive group Y in the formula (1) may be used. The polymer micelle contained in the polymer micelle solution can be used for solubilization of a fat-soluble drug or the like. As described above, the polyalkylene oxide-modified phospholipid of the present invention is close to a living cell component and further contains a high molecular weight polyalkylene oxide chain. Compared with the micelle solution obtained by using, a hydrated layer has higher stability and lower toxicity to living organisms.

Polymer micelles are used to control the target substance from 0.05 to 2
% By weight of the surfactant of the present invention in an amount of 0.2 to 8 with respect to the total amount.
What blended by weight% is preferable. The weight average particle diameter of the obtained polymer micelles by dynamic light scattering measurement is 10 to 300 n.
m, preferably 20 to 100 nm.

The emulsion is prepared by mixing the target substance to be emulsified with water and the surfactant of the present invention at 30 to 90 ° C., preferably 45 to 85 ° C., more preferably 60 to 80 ° C. The mixture is kneaded, and an aqueous phase such as water or a buffer solution is gradually added thereto and mixed to obtain an emulsion. If necessary, a known water-soluble substance can be dissolved in the aqueous phase. The preparation of the emulsion may be performed by mixing the oil phase and the aqueous phase separately as described above, or homogenizing all the components,
They may be mixed with a sonicator or the like. Usually, the target substance is 0.01 to 30% by weight, preferably 0.1 to 30% by weight based on the total amount.
10% by weight, the surfactant of the present invention is 0.1% relative to the total amount.
４０40% by weight, preferably 0.5〜１０10% by weight.

To prepare the solubilizing solution, the target substance to be dissolved in water and the surfactant of the present invention are usually mixed at room temperature to 50 ° C., preferably at 30 to 45 ° C., and then mixed with water or water such as a buffer solution. The phases are slowly added and mixed to obtain a uniform lysate. If necessary, a known water-soluble substance can be dissolved in the aqueous phase. For mixing, a homogenizer and a sonicator can be used.
Usually, the target substance is 0.01 to 10% by weight based on the total amount,
The surfactant is preferably used in an amount of 0.1 to 5% by weight, and the surfactant of the present invention is used in an amount of 0.1 to 30% by weight, preferably 1 to 10% by weight based on the total amount.

When Y in the formula (1) is a reactive functional group other than a hydrogen atom, the surfactant of the present invention can be used as an amphiphilic chemical modifier for a physiologically active substance. The amphiphilic chemical modifier may include one or more of the surfactants of the present invention, and may further include other known components used for chemical modification of a physiologically active substance. The physiologically active substance to be chemically modified is not particularly limited as long as it has a functional group such as an amino group, a carboxyl group, a hydroxyl group, and a thiol group. Examples include enzymes, antibodies, other oligos and polypeptides, proteins, sugars, lipids, glycoproteins, glycolipids, hormones, and the like.

When the surfactant of the present invention is used as an amphipathic chemical modifier for a physiologically active substance, Y in the above formula (1)
Preferred are an amino group, a carboxyl group, an aldehyde group and a thiol group. Amino group, carboxyl group,
When it has an aldehyde group and a thiol group, it can be easily reacted with a functional group present in a physiologically active substance. For example, when Y is a carboxyl group, by forming a CONH bond with an amino group in a physiologically active substance,
A polyalkylene oxide-modified phospholipid skeleton can be introduced into a physiologically active substance.

The amphiphilic chemical modifier of a physiologically active substance can be used as a solubilizing solution, an emulsion, a dispersion, or a polymer micelle solution. For example, a lipid-soluble drug is used to prepare a polymer micelle solution using the polyalkylene oxide-modified phospholipid of the present invention by the above method, and further, when Y in the above formula (1) is a reactive functional group such as a carboxyl group, an antibody is prepared. By reacting with and binding to an amino group of a protein, a polymeric micelle preparation that can be administered to a target site can be obtained.

A physiologically active substance chemically modified with the amphiphilic chemical modifier of the present invention can be used as a liposome component. In this case, a physiologically active substance can be present on the liposome surface. For example, when the physiologically active substance is an antibody protein, the physiologically active substance can be used as a ligand of the target cell, so that the liposome can be intensively transported to the target cell. When used as an amphipathic chemical modifier for a physiologically active substance, it can be used as a solubilizing solution, emulsion, dispersion, polymer micelle solution and liposome solution.

The liposome of the present invention is a liposome comprising a liposome-forming agent containing the polyalkylene oxide-modified phospholipid of the present invention. Further, other known lipids used for forming a liposome membrane may be contained as a component. Usually, it is desirable to use the polyalkylene oxide-modified phospholipid of the present invention in an amount of 0.002 to 0.2 mol, preferably 0.01 to 0.1 mol, based on 1 mol of the phospholipid. The polyalkylene oxide-modified phospholipid is preferably used in an amount of 0.1 to 20% by weight based on the total composition of the liposome.

Liposomes obtained using the liposome-forming agent of the present invention can be produced using the polyalkylene oxide-modified phospholipid of the present invention and other lipids. Examples of other lipids include phospholipids, sterols, and compounds having a saturated or unsaturated acyl group having 8 to 22 carbon atoms. The other lipid may be a phospholipid containing phosphatidylcholine alone, or may be a mixed lipid with other lipids.

The phospholipid means glycerophospholipid, sphingolipid, glyceroglycolipid, and the sterols means cholesterol, dihydrocholesterol, ergosterol, lanosterol and the like. As the glycerophospholipid, it has 4 to 24 carbon atoms, preferably 12 carbon atoms.
And phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine having -20 saturated or unsaturated linear or branched acyl groups. It can also be used as a mixed lipid body derived from natural products, such as egg yolk lecithin and soybean lecithin.

The acyl group having 4 to 24 carbon atoms includes caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, arachinic acid, behenic acid and the like. An acyl group derived from a carboxylic acid is exemplified. Other lipids may be, for example, those composed of mixed lipids having the following composition ratios: phosphatidylcholine / cholesterol / phosphatidylglycerol is 20 to 90/10 to 60/2 to 40 mol%, preferably 30 to 60 / mol. 20-50 / 15-25
Mol%.

The liposome obtained from the liposome-forming agent of the present invention can be produced by a known method. For example, as a general Bangahm method, a raw material lipid is dissolved in an organic solvent capable of dissolving, a solvent is removed using an evaporator or the like to form a lipid thin film, and after hydration emulsification at a temperature equal to or higher than the phase point transfer, an ultra-high-pressure emulsion is formed. A liposome solution can be obtained by sonication. Further, a method of hydrating the above-mentioned raw material lipid, emulsifying the lipid at a temperature equal to or higher than the phase transition point using a homogenizer or the like, and then performing pressure filtration using a polycarbonate membrane (Hope
MJ et al, Biochimica et Biophysica Acta, 812, 5
5 (1985)). Also, the obtained liposome is a multilamellar liposome,
A single lamellar liposome may be adjusted to an appropriate particle size. Preferably, it is a single lamellar liposome having a particle size of 60 to 30 nm, more preferably 90 to 200 nm.

Since the liposome-forming agent of the present invention contains the polyalkylene oxide-modified phospholipid of the present invention,
It is excellent in storage stability as a liposome solution, is low in the content of monoacylphospholipids having strong biotoxicity, and is low in the content of nitrogen-containing impurities such as triethylamine, so that it is safe for living organisms. Therefore, by using the liposome-forming agent of the present invention, liposomes that can be used as a highly safe drug delivery system can be obtained.

The following is presumed as the reason why the liposome solution has excellent storage stability. Monoacyl phospholipids have a lower hydrophobicity than diacyl phospholipids and are therefore easily detached from the liposome surface. For this reason, conventional liposomes containing a large amount of monoacylphospholipids tend to become unstable because monoacylphospholipids are desorbed from the liposome surface and the hydrated layer on the liposome surface is reduced, causing aggregation of liposome particles to cause phase separation and instability. On the other hand, since the polyalkylene oxide-modified phospholipid of the present invention has a low content of monoacyl compound, elimination of the phospholipid from the liposome surface hardly occurs. For this reason, the storage stability of the liposome solution becomes excellent.

In the method for synthesizing the polyalkylene oxide-modified phospholipid of the present invention, a nitrogen-containing base such as triethylamine is not used as a catalyst. Therefore, a harmful base derived from the catalyst does not remain in the polyalkylene oxide-modified phospholipid, and a polyalkylene oxide-modified phospholipid that is safe for a living body and has high purity can be obtained.

The polyalkylene oxide-modified phospholipid of the present invention can be easily produced by the following step (A). Step (A): a solid obtained by mixing an activated form of the polyalkylene oxide compound represented by the formula (2) and the phospholipid represented by the formula (3), wherein the aqueous solution is an alkali metal salt showing alkalinity and containing no nitrogen Reacting in an organic solvent in the presence of a salt.

Formula (2) used in step (A)
Activated polyalkylene oxide compounds and the formula
In the phospholipid represented by (3), R¹CO, R^TwoC
O, R ^ThreeO, k, n, X, p and Y are represented by the above formula (1).
Same as described.

In the polyalkylene oxide compound represented by the formula (2), Z is an activity-imparting group, and is a group that imparts the polyalkylene oxide compound with a reaction activity with the phospholipid represented by the formula (3). Yes, electron-withdrawing groups and other groups are included. Specific examples of such a group include an imidazole group, a 4-nitrophenyloxy group, a benzotriazole group, a chlorine, a methoxy group, an ethoxy group, a propyloxy group, a carbonyloxy-N-2-pyrrolidinone group, and a carbonyl-2-yl group. An oxypyrimidine group,
Examples include an N-succinimidyloxy group and a pentafluorobenzoyl group. Among these, an imidazole group, a 4-nitrophenyloxy group, a benzotriazole group, chlorine, and an N-succinimidyloxy group are preferable.
Particularly, a 4-nitrophenyloxy group is particularly preferred.

The phospholipid represented by the formula (3) may be a natural phospholipid or a synthetic phospholipid. Specific examples include natural and synthetic phosphatidylethanolamines such as soybean and soybean hydrogenated phosphatidylethanolamine, egg yolk and egg yolk hydrogenated phosphatidylethanolamine. As the phospholipid represented by the formula (3), phosphatidylethanolamine in which k is 2 is preferable.

The solid salt used in the step (A) is an alkali metal salt showing an alkaline aqueous solution and containing no nitrogen. As the solid salt, a compound capable of binding an activated form of the polyalkylene oxide compound represented by the formula (2) and the amino group of the phospholipid of the formula (3) can be used without limitation. Specifically, a solid salt is one in which the pH when dissolved in water to form an aqueous solution indicates alkalinity. Preferably, the pH is 7.1 to 13, and more preferably, the pH is 7.1 to 11. The solid salt is preferably a solid salt containing sodium or potassium. Solid salts include carbonates, phosphates, acetates and the like. In particular,
Sodium carbonate, potassium carbonate, sodium bicarbonate,
Potassium hydrogen carbonate, sodium acetate, potassium acetate and the like. Among them, preferably, sodium hydrogen carbonate,
It is sodium carbonate, and sodium carbonate is particularly preferred. The amount of the solid salt used is preferably 0.1 to 1000 times the mol of the polyalkylene oxide compound represented by the formula (2). It is preferable that the molar ratio is not more than 1000 times because the stirring is easy. The amount of the solid salt used is, in particular, 1 to
Desirably, it is 500 times mol.

The charged amount of the activated polyalkylene oxide compound of the above formula (2) and the phospholipid of the above formula (3) are set so that the molar ratio is from 1: 1 to an excess of the phospholipid. It is desirable. Specifically, the molar ratio between the activated form of the polyalkylene oxide compound and the phospholipid is 1: 1 to 1: 5, preferably 1: 1.1 to 1: 2, and more preferably 1: 1. If the phospholipids are in excess, they can be removed from the product in the steps described below. On the other hand, if the polyalkylene oxide compound becomes excessive, unreacted polyalkylene oxide compound remains after the reaction. In this case, when the molecular weight of the polyalkylene oxide compound is about 100 to about 3000, the remaining polyalkylene oxide compound can be removed by a method such as recrystallization or crystallization. However, the yield of the polyalkylene oxide-modified phospholipid may decrease. On the other hand, when the molecular weight of the polyalkylene oxide compound is about 3,000 or more, it is often difficult to remove the remaining polyalkylene oxide compound, and it becomes difficult to obtain a high-purity polyalkylene oxide-modified phospholipid. Ratios are preferred.

In the production method of the present invention, the polyalkylene oxide compound represented by the formula (2) is reacted with the phospholipid represented by the formula (3) in the presence of the solid salt as described above. be able to. However, in some cases, the polyalkylene oxide-modified phospholipid of the present invention can also be obtained by reacting an activated phospholipid with a polyalkylene oxide compound.

The organic solvent used in the reaction of the step (A) is
Any material that does not react or hardly reacts with the starting materials and reaction products can be used without particular limitation. Examples include aprotic solvents such as ethyl acetate, dichloromethane, chloroform, benzene and toluene. Of these, toluene and chloroform are preferred. An organic solvent having a hydroxyl group such as ethanol is not preferred because it reacts with the terminal active group of the activated polyalkylene oxide compound represented by the formula (2). There is no problem in reactivity with dichloromethane etc.,
Since it has a low boiling point, it is not preferable in terms of work.

The reaction temperature of the reaction of the step (A) is 30 to 90.
° C, preferably 40 to 80 ° C. The reaction time is 1 hour or more, preferably 2 to 10 hours.

By reacting the polyalkylene oxide compound represented by the above formula (2) with the phospholipid represented by the above formula (3), the polyalkylene of the present invention represented by the above formula (1) An oxide-modified phospholipid is obtained.

In the step (A), the terminal of the polyalkylene oxide compound represented by the formula (2) is an amino group, a carboxyl group or a thiol group. Preferably, it is provided. For example, in the case of an amino group, a protecting group such as a tert-butoxycarbonyl group or a 9-fluorenylmethylcarbonyl group is attached, in the case of a carboxyl group, esterified and protected by a methyl group or the like, and in the case of a thiol group, Is preferably protected with a protecting group such as St-butyl sulfide group.

After filtration of the solid salt from the reaction solution obtained in the step (A), the filtrate of the present invention represented by the above formula (1) is concentrated or crystallized by pouring it into a poor solvent. The alkylene oxide-modified phospholipid can be obtained with high purity and high yield. The filter medium used for filtration is not particularly limited as long as it can remove insolubles of the liquid to be treated. Usually, various kinds of paper such as paper having a retention particle pore diameter of 1 to 10 μm and having solvent resistance, glass, etc. A material filter can be used. The filtration method is not limited, and for example, a method such as vacuum filtration, pressure filtration, or centrifugal filtration can be used. Further, by adjusting the pH of the filtrate obtained in the step (A), the polyalkylene oxide-modified phospholipid is converted to a hydrogen type in which M in the phosphate group of the formula (1) is hydrogen and M is sodium or potassium. It can also be converted to a certain alkali salt type.

The obtained solution of the step (A) containing the polyalkylene oxide-modified phospholipid or the polyalkylene oxide-modified phospholipid is subjected to the following steps (B) to (D).
, A highly pure polyalkylene oxide-modified phospholipid can be obtained. Steps (B) to (D) are preferably performed after removing the solid salt from the reaction solution of step (A) by the above-mentioned filtration.

In the step (B), the excess phospholipid represented by the above formula (3), which is present as an insoluble substance when the crystals obtained in the step (A) are dissolved in an organic solvent, ie, unreacted phospholipid, For example, phosphatidylethanolamine is removed. As the organic solvent to be used, a solvent which dissolves the target polyalkylene oxide-modified phospholipid but does not dissolve the excess phospholipid, or a solvent having low phospholipid solubility is preferable. As the organic solvent, ethyl acetate or acetone is preferable, and acetone is particularly preferable. The dissolution temperature in the step (B) is preferably from 0 to 80C, particularly preferably from 20 to 70C. The amount of the organic solvent used is 1 to 100 times the weight of the crystal, preferably 2 to 50 times.
Weight times.

After filtering the insoluble matter from the solution in the step (B), the filtrate is concentrated or poured into a poor solvent to crystallize the polyalkylene oxide-modified phosphorus of the present invention represented by the above formula (1). Lipids can be obtained with high purity and high yield. Crystallization can be performed only by cooling the obtained filtrate, but depending on the type of the solvent, the polyalkylene oxide-modified phospholipid represented by the formula (1) does not sufficiently precipitate and remains in the solution. , The yield may decrease. Alternatively, crystallization may be performed by removing the organic solvent by distillation or the like. When the organic solvent is distilled off, it is desirable to carry out the distillation at a temperature of 80 ° C. or lower under reduced pressure. Organic solvent distillation temperature is 8
If the temperature exceeds 0 ° C., undesirable side reactions such as decomposition of the acyl group of the polyalkylene oxide-modified phospholipid represented by the formula (1) may occur.

As described above, the pH of the filtrate obtained in the step (B) can be adjusted to convert the polyalkylene oxide-modified phospholipid into a hydrogen type or an alkali salt type.

The step (B) may be performed after the step (A) or before the steps (C) and (D) described below, or may be performed in the step (C) and / or the step (D). May be performed after.

The crystals obtained in the step (B) can be directly used as the polyalkylene oxide-modified phospholipid of the present invention or can be further purified.

In the method of the present invention, after the step (A), the following steps (C) and / or (D) are carried out to obtain the starting material of the formula (2) in the step (A).
It is preferable to remove the Z-derived compound generated from the activity-imparting group Z of the activated polyalkylene oxide compound represented by Steps (C) and (D) correspond to step (A), respectively.
After the step (B), it may be before or after the step (B), and either the step (C) or the step (D) may be first. However, steps (C) and (D) are preferably performed after step (B), and step (D) is preferably performed after step (C).

In the step (C), the compound derived from the Z is removed by dissolving the crystals obtained in the step (A), cooling or adding a poor solvent to precipitate crystals of the polyalkylene oxide-modified phospholipid. For purification. The solvent used in the step (C) is a solvent in which the crystals obtained in the step (A) are dissolved and the polyalkylene oxide-modified phospholipid crystals are precipitated by cooling, or a polyalkylene oxide-modified phosphorus is added by adding a poor solvent. Solvents capable of crystallizing lipids are preferred. When crystals are precipitated, the target polyalkylene oxide-modified phospholipid crystallizes, and Z
Particularly preferred are solvents in which the compound of origin remains dissolved. The amount of the solvent used in the step (C) is 1
It is 100 times by weight, preferably 2 to 50 times by weight.

In the step (C), after recrystallization, cooling is performed or crystallization is performed using a poor solvent. Preferably, if the cooling is performed at 10 ° C. or less, crystallization is sufficiently performed, and crystals are obtained with a good yield. The solvent may be distilled off for crystallization. When the solvent is distilled off, it is desirable to carry out the distillation at a temperature of 80 ° C. or lower under reduced pressure. Organic solvent distillation temperature is 80 ℃
If it exceeds, undesirable side reactions such as decomposition of the acyl group of the polyalkylene oxide-modified phospholipid represented by the formula (1) may occur.

As a specific method of the step (C), the following method can be mentioned. (I) at least one selected from ethyl acetate and acetone
After dissolution in the seed solvent, the crystals of the polyalkylene oxide-modified phospholipid are precipitated by cooling. (Ii) at least one selected from ethyl acetate and acetone
After dissolving in a kind of solvent, polyalkylene oxide-modified phospholipid crystals are precipitated using ether or an aliphatic hydrocarbon having 5 to 8 carbon atoms. (Iii) At least one solvent selected from ethyl acetate and acetone is combined with ether or a solvent of an aliphatic hydrocarbon having 5 to 8 carbon atoms, dissolved, and then cooled to form crystals of the polyalkylene oxide-modified phospholipid. Precipitate. Among the above methods, the method of dissolving with (i) ethyl acetate, cooling, and crystallizing is particularly preferable.

The carbon number 5 to 5 used in the step (C)
The aliphatic hydrocarbon of No. 8 is not particularly limited. For example, pentane, isopentane, neopentane, hexane, isohexane, 3-methylpentane, neohexane, 2,3-
Dimethylbutane, heptane, 2-methylhexane, 3-
Methylhexane, 3-ethylpentane, 2,2-dimethylpentane, 2,3-dimethylpentane, 3,3-dimethylpentane, 2,3,3-trimethylbutane, octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethyl Hexane, 2-methyl-3-ethylpentane, 3-methyl-3-ethylpentane, 2,2,3-trimethylpentane, 2,2,4-trimethylpentane, 2,2,3,3-tetramethylbutane and the like Can be mentioned. Of these, hexane and heptane are preferred.

When it is desired to further increase the purity in the step (C), the polyalkylene oxide-modified phospholipid having a higher purity can be obtained by repeating the step (C) several times in the same manner. The crystals obtained in the step (C) can be directly used as the polyalkylene oxide-modified phospholipid of the present invention, or can be further subjected to another purification step.

In the step (D), the compound derived from Z is removed by dissolving the crystals in a solvent, adding an adsorbent, and stirring the mixture. Step (D) can also be performed using a filtrate obtained by filtering the reaction solution obtained in step (A). The solvent used in the step (D) is ethyl acetate, chloroform,
Toluene, acetone and the like are preferred, and ethyl acetate is particularly preferred.

The temperature for the treatment using the adsorbent is 10 to 85.
° C, preferably 40 to 70 ° C, for a time of 10 minutes to 5 hours,
Preferably, it is 30 minutes to 3 hours. The crystals obtained in the step (A) may be dissolved by heating and treated with an adsorbent. However, in many cases, the crystals do not dissolve at the above temperature or the viscosity of the solution is high. The treatment is preferably performed by diluting in a solvent that dissolves the alkylene oxide-modified phospholipid. If the treatment temperature is lower than 10 ° C., the polyalkylene oxide-modified phospholipid may be precipitated, and when the adsorbent is removed, the polyalkylene oxide-modified phospholipid is also removed together, which is not preferable because the yield decreases. If the temperature exceeds 85 ° C., hydrolysis of the polyalkylene oxide-modified phospholipid may occur during treatment with the adsorbent when a small amount of water is present. The amount of the adsorbent used is 0.1 to 100 parts by weight of the crystal to be treated.
It is desirably 1 to 200 parts by weight, preferably 1 to 50 parts by weight. When the amount of the adsorbent is 0.1 to 200 parts by weight, the compound derived from Z can be sufficiently and efficiently removed.

After the adsorbent is treated in the step (D) and the adsorbent is removed by a method such as filtration, the mixture can be cooled or crystallized using a poor solvent. Preferably, if the cooling is performed at 10 ° C. or less, crystallization is sufficiently performed, and crystals are obtained with a good yield. After removing the adsorbent, the solvent may be distilled off for crystallization, but when the solvent is distilled off,
It is desirable to carry out at 80 ° C. or less under reduced pressure. If the temperature at which the organic solvent is distilled off exceeds 80 ° C., undesirable side reactions such as decomposition of the acyl group of the polyalkylene oxide-modified phospholipid represented by the formula (1) may occur.

The adsorbent used in the step (D) is an alkaline earth metal oxide, an alkaline earth metal hydroxide, an adsorbent containing aluminum or silicon or activated carbon, such as aluminum hydroxide, aluminum oxide, An adsorbent containing magnesium oxide, magnesium hydroxide, silicon oxide and the like, activated carbon and the like can be mentioned. Commercially available adsorbents containing these compounds include Kyoward 200, Kyoward 300, and Kyoward 50.
0, KYOWARD 600, KYOWARD 700, KYOWARD 1000, KYOWARD 2000 (manufactured by Kyowa Chemical Industry Co., Ltd., trademark), TOMIC-AD300, TOMIC-AD500, TOMIC-AD700 (manufactured by Tomita Pharmaceutical Co., Ltd., trademark) And the like. The adsorbent can be used alone or in combination of two or more.

The crystals obtained in the step (D) can be used as the polyalkylene oxide-modified phospholipid of the present invention as they are, or the filtrate of the step (D) is further treated with an adsorbent without crystallization. For example, it can be further purified.

According to the above-mentioned production method, the content of monoacylphospholipid by-produced is small, and it is used as a catalyst in the conventional production method, and it is difficult to completely separate it from the final product. A high-purity polyalkylene oxide-modified phospholipid having a small content of impurities such as a base having a nitrogen atom remaining in the final product can be easily produced in a high yield.

[0070]

The polyalkylene oxide-modified phospholipid of the present invention is novel and useful. Since the polyalkylene oxide-modified phospholipid of the present invention is a high-purity polyalkylene oxide-modified phospholipid that is low in the content of monoacylphospholipids and impurities having a nitrogen atom and is highly safe for living bodies, it is a surfactant, especially an emulsifier. , A solubilizer, a polymeric micelle-forming agent, a dispersant, an amphiphilic chemical modifier, a liposome-forming agent, and the like. Furthermore, among these, it can be suitably used as a liposome-forming agent. In the production method of the present invention, the polyalkylene oxide compound represented by the formula (2)
The phospholipid represented by the formula (3) is reacted in an organic solvent in the presence of a solid salt that is alkaline metal salt that exhibits alkalinity when dissolved in water and does not contain nitrogen. Therefore, it is possible to easily produce a polyalkylene oxide-modified phospholipid having a low content of a monoacylphospholipid compound and a low content of impurities having a nitrogen atom in high purity and high yield.

[0071]

Embodiments of the present invention will be described below. In each case,% is% by weight unless otherwise specified.

Example 1 (1) Synthesis of monomethylpolyoxyethylenecarbamyl distearoylphosphatidylethanolamine (formula (4) shown below)

[0073]

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Toluene (80 mL) was added to polyoxyethylene monomethyl ether (molecular weight: 2,000, 20 g, 10 mmol), refluxed at 110 ° C., and dehydrated for 1 hour. After cooling to 50 ° C., 1,1′-carbonyldiimidazole (1.95 g, 12 mmol) was added and reacted for 2 hours to obtain an activated form. Next, sodium carbonate (42.4 g, 400 mmol), distearoyl phosphatidylethanolamine (11.22 g, 15 m
mol), the mixture was heated to 65 ° C., and reacted for 8 hours.
After filtration of sodium carbonate, hexane (300 m
L) was added and allowed to crystallize. After filtering the crystals (this crystal 1
g was sampled. This is referred to as a sample in the step (A): hereinafter, a sample similarly sampled is referred to as a sample), acetone (80 mL) was added to the crystal, and the mixture was heated to 50 ° C. The mixture was filtered with a glass filter to remove insolubles. Hexane (160 mL) in the filtrate
Was added, and the mixture was crystallized and cooled to 5 ° C. Thereafter, the crystals were filtered (sample in step (B)).

Ethyl acetate (400 mL) was added to the crystals,
After dissolving at 65 ° C and stirring for 30 minutes, the mixture was cooled to 5 ° C. The precipitated crystals were filtered (sample in step (C)).
Similarly, the step with ethyl acetate was performed once again (sample in step (C)). The crystals were taken up in ethyl acetate (40
0mL) and dissolved as Kyoward # 20 as an adsorbent
00 (5 g) and Kyoward # 700 (0.8 g) were added and stirred at 65 ° C. for 1 hour. After filtering the adsorbent, 5 ℃
And crystallized (sample). After filtering the crystals, the adsorbent treatment was similarly performed once. Hexane (20
0mL), and the crystals were washed, filtered and dried to obtain the target compound 1.
4.6 g (52.2% yield) was obtained (sample in step (D)). The purity of the finally obtained crystal is 98.
4%.

The analysis of the product at each stage was performed by thin layer chromatography (TLC) using a silica gel plate.
Was performed. A mixed solvent having a mixing ratio of chloroform and methanol of 85:15 by volume was used as a developing solvent, the color was developed with iodine vapor, and the contained substances were quantified by comparison with a known amount of a standard substance. Similarly, as a developing solvent, the mixing ratio of chloroform, methanol, water, and ammonia water is 6
TLC was performed using a mixed solvent of 5: 25: 4: 0.1. In the former, the lyso-form, free phospholipid, free polyethylene glycol derivative, and triethylamine can be quantified as impurities, and in the latter, the amount of triethylamine in the triethylamine salt of the phosphoric acid part in the polyoxyethylene-modified phospholipid can be quantified. . Table 1 shows the results of determining the purity by these methods.

On the other hand, in order to measure the amount of the compound derived from Z contained in the substance in each step, the samples were also analyzed. After drying the samples (1) to (5) under reduced pressure, 50 mg of each sample was placed in a sample tube, and the total amount was made up to 5 g with ethanol and dissolved. The absorbance at 248 nm derived from 1,1′-carbonyldiimidazole (CDI) was measured with a spectrophotometer. The removal rate of the compound derived from 1,1′-carbonylimidazole in each step was determined from the measured value of each sample, and the respective removal rates obtained are shown in Table 2.

From the results shown in Table 2, it was confirmed that in each step, the number of compounds derived from Z was reduced, and the polyalkylene oxide-modified phospholipid was purified. It is to be noted that although it finally contains about 0.02% of a compound derived from 1,1′-carbonyldiimidazole, this value is a value that can be ignored as the impurity content.

Example 2 (2) Synthesis of monomethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine (formula (5) shown below)

[0080]

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A reactor was charged with polyoxyethylene monomethyl ether (molecular weight: 2,000, 50 g, 25 mmol), sodium carbonate (53 g, 500 mmol) and toluene (200 mL), and heated to 75 ° C. p-Nitrophenyl chloroformate (12.6 g, 62.5 m
mol) was added and reacted for 9 hours to obtain an activated form. After cooling to 65 ° C., distearoyl phosphatidylethanolamine (28.1 g, 37.5 mmol)
l) was added thereto and reacted for 7.5 hours. After filtering the sodium carbonate, hexane (500 mL) was added to the filtrate, and the mixture was cooled to 5 ° C., and the precipitated crystals were filtered. Acetone (200 mL) was added to the crystals, the mixture was heated to 50 ° C., and then filtered with a glass filter to remove insolubles (step (B)).

After adding hexane (500 mL) to the filtrate, the mixture was cooled to 5 ° C. After the precipitated crystals were filtered, they were subjected to step (C). In the step (C), ethyl acetate (750
mL), dissolve at 65 ° C, stir for 30 minutes, and
And the precipitated crystals were filtered. Similarly, the step (C) using ethyl acetate was performed once again. The crystals were dissolved in ethyl acetate (750 mL), and Kyoward # 2000 (12 g) and Kyoward # 700 (1 g) were used as adsorbents.
Was added and stirred at 60 ° C. for 1 hour. After filtering the adsorbent, 5
C., and the precipitated crystals were filtered (step (D)).
Similarly, the treatment with the adsorbent in step (D) was performed twice.
The crystals were washed with hexane (300 mL), filtered and dried to obtain 38.2 g (yield 54.6%) of the target compound.
Purity was 99.5%. Table 1 shows the results of the purity.

Example 3 (3) Synthesis of monomethylpolyoxyethylenecarbamyl distearoylphosphatidylethanolamine (formula (6) shown below)

[0084]

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Polyoxyethylene monomethyl ether (molecular weight 5000, 20 g, 4 mmol), sodium carbonate (17.0 g, 160 mmol) and toluene (75 mL) were added, and the mixture was heated to 75 ° C. p-nitrophenyl chloroformate (2.7 g, 13.2 mmol
1) was added and reacted for 9 hours to obtain an activated form. After cooling to 65 ° C., distearoyl phosphatidylethanolamine (4.5 g, 6 mmol) was added,
The reaction was performed for 6 hours. After filtering the sodium carbonate, hexane (200 mL) was added to the filtrate, and the precipitated crystals were filtered. Acetone (150 mL) was added to the crystals, and the mixture was heated to 50 ° C., and then insolubles were removed with a glass filter (step (B)).

Hexane (300 mL) was added for crystallization. After filtration, ethyl acetate (400 mL) was added to the obtained crystals.
Was added, and the mixture was dissolved at 65 ° C., stirred for 30 minutes, cooled to 5 ° C., and the precipitated crystals were filtered (step (C)). Further, the step (C) using ethyl acetate was performed twice. After dissolving the crystals in ethyl acetate (360 mL), Kyoward # 2000 (4 g) and Kyoward # 700 are used as adsorbents.
(0.2 g) and stirred at 65 ° C. for 1 hour. After filtering the adsorbent, the mixture was cooled to 5 ° C., and the precipitated crystals were filtered (step (D)). Similarly, the treatment with the adsorbent in step (D) was performed twice. After washing the crystals with hexane (90 mL), the crystals were filtered and dried, and 15.2 g of the target compound (65.4% yield).
I got Purity was 98.4%. Table 1 shows the purity results.
Shown in

Example 4 (4) Synthesis of monomethyl polyoxyethylene carbamyl dipalmitoyl phosphatidylethanolamine (formula (7) shown below)

[0088]

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Polyoxyethylene monomethyl ether (molecular weight 5000, 20 g, 4 mmol), sodium carbonate (33.9 g, 320 mmol) and toluene (75 mL) were added, and the mixture was heated to 75 ° C. p-Nitrophenyl chloroformate (2.82 g, 14 mmol)
Was added and reacted for 9 hours to obtain an activated form. After cooling to 65 ° C., dipalmitoyl phosphatidylethanolamine (3.2 g, 6 mmol) was added and reacted for 5 hours. After filtering the sodium carbonate, hexane (200 mL) was added to the filtrate, and the mixture was cooled to 5 ° C. The precipitated crystals were filtered to obtain crystals. Acetone (150m
L) was added, and the mixture was heated to 50 ° C., and insolubles were removed with a glass filter (step (B)).

Hexane (300 mL) was added to the filtrate for crystallization. After filtration, ethyl acetate (400
mL), dissolved at 65 ° C., and stirred for 30 minutes. 5
After cooling to ℃, the precipitated crystals were filtered (step (C)).
Further, the step (C) using ethyl acetate was performed twice. The crystals were dissolved in ethyl acetate (360 mL), and Kyoward # 2000 (4 g) and Kyoward # 7 were used as adsorbents.
00 (0.2 g) was added. After filtering the adsorbent, the mixture was cooled to 5 ° C., and the precipitated crystals were filtered (step (D)). Similarly, the treatment with the adsorbent in step (D) was performed twice. After washing the crystals with hexane (90 mL), the crystals were filtered and dried to obtain 15.2 g (yield: 65.6%) of the target compound. Purity 9
8.5%. Table 1 shows the results of the purity.

Comparative Example 1 (1) Synthesis of pyridyldithiopropionoylamino polyethylene glycol distearoylphosphatidylethanolamine Pyridyldithiopropionoylamino polyethylene glycol succinimidyl carbonate (1 g, 0.1 g)
42 mmol) in chloroform (10 mL)
Distearoyl phosphatidylethanolamine (0.
36 g, 0.44 mmol), followed by triethylamine (0.33 mL, 2.37 mmol). The reaction mixture was stirred at 40 ° C. for 10 minutes. The reaction solution was concentrated under reduced pressure using an evaporator, and then acetonitrile (50 mL)
Was added and the solution was refrigerated at 4 ° C. overnight. Next, centrifugation was performed to separate a clear solution. After the solution was concentrated by an evaporator, the crystals were dried. The yield was 1.15 g. The yield was 90.8%. Table 3 shows the results of the purity.

The free triethylamine content and free salt content shown in Table 3 can be quantified up to 5% in a thin layer chromatography (TLC) spot, but no quantification is possible beyond that. . Therefore, those exceeding 5% were set to> 5.

Comparative Example 2 (2) Synthesis of monomethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine Polyoxyethylene monomethyl ether (molecular weight: 200
(0, 20 g, 10 mmol) was added with benzene (75 mL), refluxed and dehydrated. Add carbonyldiimidazole (1.78 g, 11 mmol) and reflux for 2 hours,
An activated form was obtained. Distearoylphosphatidylethanolamine (7 g, 9.36 mmol) and triethylamine (3.1 mL, 22 mmol) were added, and the mixture was refluxed for 20 hours. The reaction solution was concentrated by an evaporator to obtain a crude product. This crude product was purified by C-18 reverse phase silica gel chromatography. The C-18 reverse phase silica gel used was (Bio-Gel A-1.5 m), and the eluent used was ethanol / water = 4/1. The yield is 5.0 g,
Yield was 18%. Table 3 shows the results of the purity.

Comparative Example 3 (3) Monomethylpolyoxyethylenecarbamyl 1-
Synthesis of palmitoyl-2-oleoylphosphatidylethanolamine Polyoxyethylene monomethyl ether (molecular weight 200
0, 5 g, 2.5 mmol) was dissolved in chloroform / toluene = 50/2 (wt / wt) (25 mL), and triphosgene (0.89 g, 3 mmol) was added, followed by a reaction at 40 ° C. for 3 hours. To this solution was added diethyl ether (7
5 mL) and precipitated. The precipitate was filtered and dried to obtain monomethyl polyoxyethylene chloroformate (4.7 g, 91%). Monomethyl polyoxyethylene chloroformate (4 g, 1.94 mmol)
l) and 1-palmitoyl-2-oleoylphosphatidylethanolamine (1.65 g, 2.3 mmol)
l) was dissolved in chloroform (20 mL), triethylamine (0.39 mL, 2.76 mmol) was added, and 6
The reaction was performed at 0 ° C. for 3 hours. After filtering off insolubles, the reaction solution was concentrated using an evaporator. Water (20 mL) was added to the residue to dissolve it, and 0.1 N hydrochloric acid (5.0 mL) was added to make the aqueous solution acidic. Methylene chloride (25 mL) in aqueous solution
Was added and extracted. Similarly, extraction was repeated twice, and sodium sulfate (30 g) was added to the combined extract and dehydrated. After the sodium sulfate was filtered, it was concentrated with an evaporator. The residue was purified by silica gel column chromatography to obtain 1.5 g of the desired compound. 28% yield
Met. Table 3 shows the results of the purity.

Comparative Example 4 (4) Synthesis of t-butoxycarbonylhydrazide polyoxyethylene succinyl phosphatidylethanolamine t-butoxycarbonylhydrazide polyoxyethylene succinimidyl carbonate (1 g, 0.42 m
mol) was dissolved in chloroform (6 mL). Distearoyl phosphatidylethanolamine (0.292
g, 0.39 mmol) and triethylamine (0.
144 mL, 1.04 mmol) and 10
Allowed to react for minutes. Acetic acid (0.06 mL, 1.0
5 mmol), and the mixture was concentrated using an evaporator. Water (7.5 mL) was added to the residue, and after dissolution, a small amount of chloroform was distilled off using an evaporator. The reaction solution is Spec
trapor CE dialysis tubing (MWCO 30
10,000 mM) and 50 mM physiological saline (1500 mL x
Dialysis was performed at 4 ° C. in 3) (8-16 hours / dialysis once). Further, the solution was dialyzed against ion-exchanged water and then filtered through a 0.2 μm sterilizing filter. After lyophilization, the desired product was obtained. The yield was 0.55 g and the yield was 43.3%. Table 3 shows the results of the purity.

[0096]

[Table 1]

[0097]

[Table 2]

[0098]

[Table 3]

Notes to Tables 1 and 3 * 1 Purity 1: Purity of the polyalkylene oxide-modified phospholipid represented by the formula (1). (Unit%) * 2 Lyso-form content: content of monoacyl phospholipid.
(Unit%) * 3 Free PE content: content of unreacted phospholipid.
(Unit%) * 4 Free PEG derivative content: Content of polyethylene glycol and its derivatives. (Unit%) * 5 Free triethylamine content: Triethylamine content. (Unit%) * 6 Triethylamine content in free salt: Triethylamine content in triethylamine phosphate salt. (unit%)

From the results shown in Tables 1 and 3, it can be seen that the production method of the example has a lower content of impurities such as lyso-form and triethylamine and produces polyalkylene oxide-modified phospholipid with higher purity than the comparative example. You can see that you can do it.

Example 5 Monomethyl polyoxyethylene carbamyl of Example 3
An emulsion was made using distearoyl phosphatidylethanolamine. That is, of the base material having the composition shown in Table 4, the oil phase portion containing the emulsifier was heated to 60 ° C. and uniformly dissolved, and then the aqueous phase portion was added at the same temperature while stirring.

[0102]

[Table 4]

After storing the prepared emulsion at 40 ° C. for one month, the emulsified state was evaluated according to the following criteria. Table 6 shows the results
Shown in 3: Stable state 2: Slightly uneven state 1: Creaming or separated state

The emulsion immediately after the preparation was applied to the skin and subjected to a sensory test. The sensory evaluation was performed by five specialized panelists. As an evaluation method, a sample was applied after the upper arm was washed, and skin irritation immediately after application and after one night had passed was evaluated according to the following three-level evaluation. Table 6 shows the total score of the five players. 3: Normal. There is no abnormality at all. 2: There is discomfort. I feel a little itching. 1: There is itching. Redness is observed on the skin.

Example 6 Monomethyl polyoxyethylene carbamyl of Example 2
A cream was made using distearoyl phosphatidylethanolamine. That is, the oil phase portion containing the emulsifier of the base material having the composition shown in Table 5 was heated to 60 ° C. and uniformly dissolved, and then the aqueous phase portion was added at the same temperature while stirring.

[0106]

[Table 5]

The prepared cream was evaluated in the same manner as in Example 5. Table 6 shows the results.

Comparative Examples 5 and 6 Monomethylpolyoxyethylenecarbamyl of Comparative Example 3
The same tests as in Examples 5 and 6 were performed using distearoyl phosphatidylethanolamine. Table 6 shows the results.

[0109]

[Table 6]

Example 7 Evaluation of stability of liposome solution Dipalmitoyl phosphatidylcholine (1.92 g,
2.64 mmol), cholesterol (0.45 g,
1.32 mmol), monomethylpolyoxyethylene carbamyl distearoyl phosphatidylethanolamine obtained in Example 2 (0.42 g, 0.15 mmol)
Was placed in an eggplant-shaped flask, dissolved by adding 50 mL of chloroform, and the solvent was removed by a rotary evaporator to form a lipid thin film on the inner wall of the flask. The solvent was sufficiently removed under reduced pressure, and a phosphate buffered saline solution of pH 7 was added.
mL was added and dispersed, and the mixture was further treated for 5 minutes with an ultrasonic cleaner to obtain a liposome solution. The obtained liposome solution was left at room temperature for one month. One month later, the dispersion state of the liposome solution was not visually observed, and was a uniform liposome solution.

Comparative Example 7 The same evaluation as in Example 7 was performed using the monomethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine obtained in Comparative Example 2. As a result, the liposome solution was not uniform, and the lipid particles had settled.

Example 8 Modification of asparaginase using carboxymethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine Carboxymethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine obtained in the same manner as in Example 3 (5 g, 0 .86 mmol) was dissolved in 50 mL of chloroform, N-hydroxysuccinimide (0.15 g, 1.29 mmol) was added, and dicyclohexylcarbodiimide (0.27 g, 1.29 mmol) dissolved in a small amount of chloroform was added. Stirred at room temperature for 2 hours. Thereafter, diethyl ether was added to the solution from which insoluble matter was removed by filtration, and the precipitated crystals were obtained by filtration. The solvent was removed under reduced pressure and used for the next step.

0.1 g of asparaginase was dissolved in 50 mL of a phosphate buffered saline solution having a pH of 7.4, and the succinimidyl carboxymethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine obtained above was added thereto. For 4 hours. The reaction solution was dialyzed against a phosphate buffered saline solution having a pH of 7.4 and 4 ° C. to remove unreacted substances, and then freeze-dried to obtain carboxymethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine. A dried product bound to asparaginase could be obtained.

Example 9 Preparation of Polymeric Micellar Solution of Soy Hydrogenated Phosphatidylcholine Using Carboxymethyl Polyoxyethylene Carbamyl Distearoylphosphatidylethanolamine Soybean hydrogenated phosphatidylcholine (0.1 g, 0.13 m
mol) and carboxymethyl polyoxyethylene carbamyl distearoyl phosphatidylethanolamine (1 g, 17 mmol) obtained in the same manner as in Example 3 were added to distilled water (5 mL) and mixed with stirring. Distilled water (95 mL) was gradually added to the homogeneous mixed solution and stirred to obtain a transparent and uniform polymer micelle solution. About the obtained solution, a particle size analyzer (NICOMP Model 370: manufactured by Nozaki Sangyo Co., Ltd.)
Was used to measure the particle size distribution. As a result, the average particle size was 35.
nm. The obtained Takako micelle solution was left at room temperature for one month. The state of the polymer micelle solution after one month is
No change was observed visually, and the polymer micelle solution was homogeneous without any precipitate.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/34 A61K 47/34 47/48 47/48 B01F 17/14 B01F 17/14 17/42 17 / 42 // C07F 9/10 C07F 9/10 A (72) Inventor Chika Ito 103-203, Toko Market, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture (72) Inventor Shunsuke Ohashi 3-32-6, Shiroyuri, Izumi-ku, Yokohama-shi, Kanagawa Prefecture ( 72) Inventor Toru Yasukochi 1-1-3 -401 F-term (reference) 1-1-3 Chigasakihigashi, Tsuzuki-ku, Yokohama-shi, Kanagawa 4C076 AA12 AA16 AA19 AA30 CC29 DD09E DD09F DD63 DD63E DD63F DD70 EE23E EE23F EE59 FF15 FF16 GG45 GG46 4C082 AC022 AC AC182 AC242 AC402 AC422 AC901 AC902 AD662 BB01 CC01 DD23 DD31 DD45 FF01 4D077 AA04 AB12 AC01 DD29Y DE02Y DE06Y DE09Y DE10Y DE16Y DE18Y DE24Y DE32Y 4H050 AA01 AA02 AA03 AB20 AB68 AC70 AD15 AD17 BA02 BA32 BB11 BB12 BB16 BB17 4J005 AA04 BD00 BD05 BD07

Claims (22)

[Claims] 1. A polyalkylene oxide-modified phosphorus represented by the following formula (1) having a monoacylphospholipid content of 3% by weight or less and a nitrogen-containing base content of 0.02% by weight or less. Lipids. Embedded image (In the formula (1), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, k is 1 to 6, R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is a carbon atom. The average number of moles of the oxyalkylene group represented by the formulas 2 to 4 is 10 to 800. M is a hydrogen atom, sodium or potassium, X
Is a divalent hydrocarbon group having 1 to 3 carbon atoms or -C (= O)
(CH ₂ ) q- (where q is 1 to 4), and p is 0 or 1. When p is 0, Y is a hydrogen atom or a group having 1 to 4 carbon atoms.
Is an alkyl group. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. ) 2. The polyalkylene oxide-modified phospholipid according to claim 1, wherein the content of the monoacyl phospholipid is 2% by weight or less. 3. The polyalkylene oxide-modified phospholipid according to claim 1, wherein p is 0, Y is a methyl group, and the content of the monoacyl phospholipid is 0.5% by weight or less. 4. A method for producing a polyalkylene oxide-modified phospholipid having the following step (A). Step (A): A solution containing an activated form of a polyalkylene oxide compound represented by the following formula (2) and a phospholipid represented by the following formula (3), wherein the aqueous solution is an alkali metal salt showing alkalinity and containing no nitrogen. Reacting in an organic solvent in the presence of a salt. Embedded image (In the formula (2), R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, n is an average number of added moles of the oxyalkylene group having 2 to 4 carbon atoms, and is a number of 10 to 800. X is a carbon number. 1-3
Divalent hydrocarbon group or _{-C (= O) (CH 2} ) q- of (here q 1 to 4), p is 0 or 1. When p is 0, Y is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. Z is an activity imparting group. ) (In the formula (3), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, and k is 1 to 6.) 5. The method according to claim 1, wherein R ¹ CO and R ² CO have 12 to 2 carbon atoms.
The method according to claim 4, wherein the acyl group is 0. 6. The method according to claim 4, wherein p is 0 and Y is a methyl group. 7. The method according to claim 4, wherein the solid salt used in step (A) is sodium carbonate, and the organic solvent is toluene or chloroform. 8. The production method according to claim 4, comprising the following step (B) after the step (A). Step (B): a step of removing unreacted phospholipid represented by the formula (3) using ethyl acetate or acetone. 9. The method according to claim 4, comprising the following step (C) after the step (A). Step (C): a step of performing recrystallization using ethyl acetate and / or acetone as a solvent. 10. In the step (C), further having 5 carbon atoms
The production method according to claim 9, wherein at least one compound selected from the group consisting of aliphatic hydrocarbons and ethers of Nos. To 8 is used as a solvent. 11. The method according to claim 9, wherein the step (C) is performed after the step (B). 12. The method according to claim 4, further comprising the following step (D) after the step (A). Step (D): a step of performing purification using an adsorbent. 13. The method according to claim 12, wherein the adsorbent used in the step (D) is an alkaline earth metal oxide, an alkaline earth metal hydroxide, an adsorbent containing aluminum or silicon, or activated carbon. Method. 14. The method according to claim 12, wherein the step (D) is performed after the step (B). 15. The following formula (1) wherein the polyalkylene oxide-modified phospholipid has a monoacylphospholipid content of 3% by weight or less and a nitrogen-containing base content of 0.02% by weight or less. The method according to any one of claims 4 to 14, which is a compound represented by the formula: Embedded image (In the formula (1), R ¹ CO and R ² CO are each independently an acyl group having 4 to 24 carbon atoms, k is 1 to 6, R ³ O is an oxyalkylene group having 2 to 4 carbon atoms, and n is a carbon atom. The average number of moles of the oxyalkylene group represented by the formulas 2 to 4 is 10 to 800. M is a hydrogen atom, sodium or potassium, X
Is a divalent hydrocarbon group having 1 to 3 carbon atoms or -C (= O)
(CH ₂ ) q- (where q is 1 to 4), and p is 0 or 1. When p is 0, Y is a hydrogen atom or a group having 1 to 4 carbon atoms.
Is an alkyl group. When p is 1, Y is a hydrogen atom, an amino group, a carboxyl group, an aldehyde group, a glycidyl group or a thiol group. ) 16. A surfactant comprising the polyalkylene oxide-modified phospholipid according to claim 1. Description: A liposome-forming agent comprising the polyalkylene oxide-modified phospholipid according to any one of claims 1 to 3. 18. A liposome comprising the polyalkylene oxide-modified phospholipid according to claim 1. 19. An amphipathic chemical modifier for a physiologically active substance, comprising the polyalkylene oxide-modified phospholipid according to claim 1. Description: 20. An emulsifier for a physiologically active substance, comprising the polyalkylene oxide-modified phospholipid according to claim 1. 21. A solubilizing agent for a physiologically active substance, comprising the polyalkylene oxide-modified phospholipid according to claim 1. A polymer micelle comprising the polyalkylene oxide-modified phospholipid according to any one of claims 1 to 3.