JPH092975A - Reactive endoplasmic reticulum-forming agent and reactive endoplasmic reticulum - Google Patents

Abstract

PURPOSE: To obtain a reactive endoplasmic peticulum-forming agent containing a specific reactive cholesterol derivative, capable of simply and efficiently immobilizing various functional substances through a (poly)oxyalkylene chain to the tip of an endoplasmic reticulum by a covalent bond. CONSTITUTION: This reactive endoplasmic reticulum-forming agent contains a reactive cholesterol derivative of formula I (OA is a 2-4C oxyalkylene; (n) is the average number of additions mols of OA and is 1-1000; R is H or methyl; X is a bivalent organic residue; (p) is 0 or 1). In order to obtain a compound of formula I in which (p) is 0, for example, an alkylene oxide is subjected to addition polymerization to a hydroxyl in a cholesterol of formula II in the presence of a catalyst such as NaOH, etc., in a solvent such as toluene, etc., at 0-150 deg.C for 30 minutes to 200 hours to give a (poly)oxyalkylene additive to the cholesterol. The hydroxyl group at the end of the additive is made into an N-oxycarbonylimidazole with an N,N'-carbonyldiimidazole of formula III or its substituted substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reactive endoplasmic reticulum forming agent and a reactive endoplasmic reticulum containing the same as an endoplasmic reticulum forming component. More specifically, vesicles such as drug carriers for drugs, test agents, diagnostic agents, sensors, immobilized catalysts, bioreactors, bioelectronic elements, microcapsule substitutes, cosmetic raw materials, and various functional liposomes or fat emulsions. The present invention relates to a reactive endoplasmic reticulum forming agent for use in the production of the same and a reactive endoplasmic reticulum containing the same as an endoplasmic reticulum forming component.

[0002]

2. Description of the Related Art A liposome is an endoplasmic reticulum composed of a bilayer membrane of phospholipid, and its application has been tried in various fields. In particular, application to drug carriers, sensors for diagnosis / detection, etc. has been attracting attention. Immobilizing a functional substance on the liposome surface or in the membrane to give various functions, and to measure the blood concentration of liposomes. Maintenance is a major issue.

Conventionally, regarding the immobilization of the functional substance on the surface of the liposome or in the membrane, γ-maleimidobutyloxy was added to the aminoethylcarbamylmethyl group substituted on the polysaccharide on the surface of the liposome coated with the pullulan derivative. A method of binding an antibody fragment via succinimidyl (Biochem. Biophys. Acta., 898 , 323 (1987)), or a glycolipid was added to the liposome membrane-forming component in advance, and periodate was added after liposome formation. There is a method of oxidizing and immobilizing the generated aldehyde group by reacting with an antibody (J. Biol. Chem., 255 , 10509 (1980)).

However, in these conventional methods, it is necessary to carry out a multi-step chemical reaction on the surface of the liposome membrane after liposome preparation, which limits the amount of the target functional substance to be introduced to a low level, and also depends on the reaction. There is a problem that by-products and impurities are mixed and damage to the liposome membrane is large.

On the other hand, it has been pointed out that when liposomes are administered in vivo, most of them are trapped in reticuloendothelial organs such as the liver and spleen, so that sufficient effects cannot be obtained (Cancer Res., 43 , 5328 (1983)). Therefore, as a method for improving the problem of being trapped in the reticuloendothelial system and the problem of low stability such as disintegration and aggregation of the liposome itself, a polyethylene glycol chain is introduced on the surface of the liposome. Has been attempted (for example, WO90 / 3484, Japanese Patent Laid-Open No. 1-249717).
Issue, FEBS letters, 268 , 235 (1990)). Further, it has been revealed that liposomes modified with polyethylene glycol can maintain the concentration in blood for a long period of time (Biochem. Biophys. Acta., 1066 , 29-36 (1991)).
However, since the liposome having a polyethylene glycol chain introduced by such a method does not react with the functional substance, the functional substance cannot be immobilized on the surface of the liposome.

Further, in Japanese Patent Laid-Open No. 4-346918,
First, a liposome having a maleimide group is reacted with a protein having a thiol group (thiolated protein), and then a compound having a polyalkylene glycol (thiolated polyalkylene glycol) moiety having a thiol group added to a residual maleimide group is reacted. Due to
It has been described that drug-containing antibody-bound liposomes with improved uptake in reticuloendothelial organs are obtained. However, this liposome has a problem that the expected effect cannot be sufficiently obtained because the antibody is hidden under the polyalkylene glycol layer and the reaction with the antigen at the target site is hindered.

Further, in JP-A-5-508388, α-
A liposome preparation comprising a polyoxyethylene derivative having an anion group represented by stearyl-ω-propionic acid-polyoxyethylene is disclosed. However, since the hydrophobic portion of this polyoxyethylene derivative is a monoalkyl group, it is easily detached from the liposome membrane,
Therefore, there is a problem that liposomes containing such a polyoxyethylene derivative as a film-forming component are inferior in long-term stability.

Further, in JP-A-1-249798, 2,4-bis (o-methoxypolyethylene glycol) in which a polyethylene glycol chain is introduced into cholesterol is disclosed.
It is described that the stability of the liposome is improved by using the -6-cholesteryl-s-triazine derivative as a membrane-forming component of the liposome. However, immobilization of functional substances and the like is not considered in this derivative.

[0009]

In order to solve the above problems, the object of the present invention is to introduce a (poly) oxyalkylene chain into liposomes and other vesicles, and to stabilize it for a long period of time without elimination. Reactive endoplasmic reticulum-forming agent capable of immobilizing various functional substances on the tip of a (poly) oxyalkylene chain easily and efficiently by covalent bond, and forming the endoplasmic reticulum with this forming agent. It is to provide a reactive endoplasmic reticulum containing as a component.

[0010]

The present invention is the following reactive endoplasmic reticulum-forming agent and reactive endoplasmic reticulum. (1) A reactive endoplasmic reticulum forming agent comprising a reactive cholesterol derivative represented by the following general formula (1).

Embedded image [In the Formula, OA is a C2-C4 oxyalkylene group, n
Is the average number of moles of oxyalkylene groups added, which is from 1 to 100.
Represents a positive number of zero. When n is 2 or more, the oxyalkylene groups may be the same or different and may be added randomly or in a block. R
Represents a hydrogen atom or a methyl group, X represents a divalent organic residue, and p represents 0 or 1. (2) A reactive endoplasmic reticulum comprising one or more of the reactive endoplasmic reticulum forming agent according to (1) above as an endoplasmic reticulum forming component.

In the present invention, "(poly) oxyalkylene" means "oxyalkylene and / or polyoxyalkylene" and "(poly) alkylene" means "alkylene and / or polyalkylene". In the present invention, the endoplasmic reticulum is a particle having a structure in which the reactive cholesterol derivative represented by the general formula (1) or the hydrophilic groups of the reactive cholesterol derivative and other vesicle-forming components are oriented toward the aqueous phase at the interface. Means Specific examples thereof include liposomes, which are closed vesicles composed of a bilayer membrane, fat emulsions obtained by emulsifying a mixture of vegetable oil and phospholipid, or micelles.

The oxyalkylene group represented by OA of the general formula (1) is an oxyalkylene group having 2 to 4 carbon atoms, and is an oxyethylene group, an oxypropylene group, an oxytrimethylene group or an oxy-1-ethylethylene group. , Oxy-1,2-dimethylethylene group, oxytetramethylene group and the like. These oxyalkylene groups are
Ethylene oxide, propylene oxide, oxetane,
It is a group obtained by addition-polymerizing an alkylene oxide such as 1-butene oxide, 2-butene oxide or tetrahydrofuran.

In the general formula (1), n is a positive number of 1 to 1000, preferably 10 to 300, more preferably 20 to 120. When n is 2 or more, the types of the oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks.

In the case of imparting hydrophilicity, ethylene oxide alone is preferably added as OA, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the (poly) oxyalkylene chain, the number of added moles other than ethylene oxide is increased.

In the general formula (1), when p is 1, X
The divalent organic residue represented by is not particularly limited, and examples thereof include the following organic residues.

Embedded image

The reactive cholesterol derivative in which p is 0 in the general formula (1) can be easily produced, for example, by the following two-step reaction. First, in the reaction of the first step, alkylene oxide is added to the hydroxyl group in cholesterol for addition polymerization to produce a (poly) oxyalkylene adduct of cholesterol (hereinafter abbreviated as Chol-OA). At this time, the charged molar ratio of cholesterol and alkylene oxide is selected according to the number of repetitions of the target (poly) oxyalkylene, and for example, 1: 1 to 1:
It is desirable to set it to 10,000, preferably 1:10 to 1: 300. The reaction uses metallic sodium, metallic potassium, sodium hydroxide, potassium hydroxide or the like as a catalyst, and an organic solvent such as toluene, benzene, chloroform or carbon tetrachloride as a solvent at 0 to 150 ° C. for 30 minutes.
It can be carried out by stirring for a minute to 200 hours.

Next, in the second step reaction, the hydroxyl group at the terminal of Chol-OA obtained in the first step reaction is converted to N,
N'-carbonyldiimidazole or a substitution product thereof is used for N-oxycarbonylimidazolization. At this time, the charged molar ratio of Chol-OA and N, N'-carbonyldiimidazole or a substitution product thereof is 1: 0.5-.
It is desirable that the ratio is 1:10, preferably 1: 0.9 to 1: 2. The reaction is solvent-free, or benzene, toluene, chloroform, carbon tetrachloride, acetonitrile, ethyl acetate, dimethylformamide, dimethylacetamide,
Using an organic solvent such as dimethyl sulfoxide, -100
To + 150 ° C., preferably −50 to + 80 ° C., more preferably 0 to 40 ° C., and stirring can be performed for 1 minute to 200 hours, preferably 30 minutes to 6 hours.

The reaction formula when ethylene oxide is used as the alkylene oxide is shown in the following formula (2). In the formula, n and R have the same meanings as described above.

Embedded image

The reactive cholesterol derivative in which p is 1 in the general formula (1) can be produced, for example, by the following method. 1) Cholesterol is reacted with N, N'-carbonyldiimidazole or a substituted product thereof, followed by reaction with (poly) alkylene glycol or a (poly) alkylene glycol derivative having an amino group at one end, and N, N A method of reacting ′ -carbonyldiimidazole or a substituted product thereof. 2) Cholesterol is reacted with succinic anhydride, glutaric anhydride, etc., and then with (poly) alkylene glycol or a (poly) alkylene glycol derivative having an amino group at one end, and N, N′-carbonyldiene A method of reacting imidazole or a substitution product thereof. 3) A method in which a (poly) alkylene glycol derivative having a carboxyl group at one end is dehydrated and condensed with cholesterol, and then N, N′-carbonyldiimidazole or a substituted product thereof is reacted. 4) After reacting cholesterol with N, N′-carbonyldiimidazole, an amino acid such as glycine, alanine, valine, or 4-aminobutanoic acid or 6-aminohexanoic acid is reacted, and (poly) alkylene glycol or A (poly) alkylene glycol derivative having an amino group at one end is reacted, and then N, N '
A method of reacting carbonyldiimidazole or a substitution product thereof. 5) After reacting cholesterol with hexamethylene diisocyanate, phenylene diisocyanate, xylene diisocyanate, etc., it is reacted with (poly) ethylene glycol or a (poly) alkylene glycol derivative having an amino group at one end, and then A method of reacting with N, N'-carbonyldiimidazole or a substitution product thereof. 6) After reacting cholesterol with N, N′-carbonyldiimidazole, an excess amount of diaminoalkylene such as 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane is reacted, and further, A (poly) alkylene oxide derivative having a carboxyl group at one end and a hydroxyl group at the other end or a (poly) alkylene glycol derivative activated at one end with an oxycarbonylimidazole group is reacted, and then N, N'-
A method of reacting carbonyldiimidazole or a substituted product thereof. 7) After reacting cholesterol with N, N′-carbonyldiimidazole, an excess amount of 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, or another diaminoalkylene is reacted, and further, A method of reacting a (poly) alkylene glycol derivative whose both terminals are activated with an oxycarbonylimidazole group or a substitution product thereof.

After the reactive cholesterol derivative represented by the general formula (1) is produced as described above, the obtained reaction mixture is used as it is, or after distillation, recrystallization, reprecipitation, adsorbent treatment, column treatment, After being purified and isolated by gel filtration, ultrafiltration, dialysis, ion exchange, thin layer chromatography, etc., it can be used as the reactive vesicle forming agent of the present invention.

The reactive endoplasmic reticulum forming agent of the present invention contains a reactive cholesterol derivative represented by the general formula (1), and is composed of one kind of the reactive cholesterol derivative represented by the general formula (1). , Or two or more kinds, or one or more kinds of the reactive cholesterol derivative represented by the general formula (1) and other endoplasmic reticulum-forming components capable of forming endoplasmic reticulum, for example, soybean lecithin , Egg yolk lecithin, other phospholipids, cholesterol, intralipid (trademark of Otsuka Pharmaceutical Co., Ltd.), a mixture with soybean oil, safflower oil and the like, and the like.

The reactive cholesterol derivative represented by the general formula (1) is an oxycarbonylimidazole group having high reactivity with a functional group such as an amino group, a hydroxyl group or a thiol group, particularly a primary amino group, or a substitution thereof. Since it has a body, it reacts easily with a functional substance having such a functional group to form a covalent bond. Therefore, by using the reactive vesicle-forming agent of the present invention as an vesicle-forming component, it is possible to introduce a (poly) oxyalkylene chain into the formed vesicle and to impart reactivity to the functional group. it can.

The reactive endoplasmic reticulum of the present invention is an endoplasmic reticulum containing the above-mentioned reactive endoplasmic reticulum forming agent as an endoplasmic reticulum forming component. The vesicle-forming component may contain other vesicle-forming components capable of forming vesicles in addition to the reactive vesicle-forming agent. Examples of other vesicle-forming components capable of forming vesicles include those similar to other vesicle-forming components that can be mixed with the reactive cholesterol derivative represented by the general formula (1) in the reactive vesicle-forming agent. I can give you something. The reactive endoplasmic reticulum of the present invention can be produced by a known method using these components. INDUSTRIAL APPLICABILITY The reactive endoplasmic reticulum of the present invention utilizes a oxycarbonylimidazole group or a substitution product thereof in the reactive cholesterol derivative represented by the general formula (1) as a functional group to introduce various functional substances by covalent bond. be able to.

Next, the reactive endoplasmic reticulum of the present invention will be described in detail by dividing into reactive liposomes, reactive fat emulsions and reactive micelles. A reactive liposome, which is a typical reactive vesicle, contains the reactive vesicle-forming agent of the present invention, that is, the reactive cholesterol derivative represented by the general formula (1), as a membrane-forming component (vesicle-forming component). It is a thing. The content of the reactive cholesterol derivative represented by the general formula (1) is 0.001 to 40 mol%, preferably 0.5 to 30 mol% with respect to the total amount of the reactive cholesterol derivative and other film forming components. Is desirable. If it is less than 0.001 mol%, the expected effect becomes small, and therefore it is not generally used. The reactive cholesterol derivative represented by the general formula (1) may contain one kind or two or more kinds.

As the other film-forming component used by mixing with the reactive cholesterol derivative represented by the general formula (1), those conventionally used as the film-forming component of liposomes can be used without limitation. Specifically, diphosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, soybean lecithin, egg yolk lecithin,
Phospholipids such as phosphatidylcholine and phosphatidylglycerol and polymerizable phospholipids having an unsaturated group in the fatty acid moiety; sulfoxyribosyl diglyceride, digalactosyl diglyceride, lactosyl diglyceride and other glycolipids; cholesterol and other nonpolar lipids; , Nonionic surfactant, phosphatidyl polyethylene glycol, "Biochem. Biophys. Acta., 1066 , 29-36 (199
1), the reaction product of phosphatidylethanolamine with α-hydro-ω-hydroxy-polyoxyethylene, and a mixture thereof.

The reactive liposome of the present invention contains the reactive cholesterol derivative represented by the general formula (1) and other film forming components such as lecithin, other phospholipids and cholesterol, which are used as necessary, in an organic solvent or the like. Dissolve in a suitable solvent, extrude method, vortex mixer method, ultrasonic method, surfactant removal method, reverse layer evaporation method, ethanol injection method, pre-vesicle method, French press method, W / O / W emulsion method, It can be produced by liposome formation by various known methods such as an annealing method and a freeze-thaw method. By selecting these production methods, reactive liposomes having various sizes and morphologies such as multilamellar liposomes, small unilamellar vesicles, and large unilamellar vesicles can be produced.

The thus-obtained reactive liposome has an oxycarbonylimidazole group or a substitution product thereof bound to the inner and outer surfaces of the liposome membrane via a spacer composed of (poly) oxyalkylene. A functional substance having a functional group such as a hydroxyl group or a thiol group, particularly a primary amino group, can be efficiently and easily bonded onto the bilayer membrane of a liposome via a spacer composed of (poly) oxyalkylene, and a urethane bond or a carbonate bond. Alternatively, it can be chemically immobilized by a thiocarbonate bond or the like.

Examples of the functional substance that can be immobilized on the reactive liposome include functional substances having functional groups such as amino groups, hydroxyl groups and thiol groups, or functional substances into which these functional groups are introduced. Among these, functional substances having or having a primary amino group are preferable. Specifically, for example, labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds, chemiluminescent compounds, and electrode sensitive compounds; external stimulus responsiveness such as photoresponsive compounds, pH responsive compounds, and thermoresponsive compounds. Compounds; enzymes, antibodies, other proteins, sugars, lipids, glycoproteins, glycolipids,
Examples include physiologically active substances such as hormones; drugs such as medicines and pesticides.

The immobilization reaction of the functional substance on the reactive liposome is carried out by using an aqueous solvent such as physiological saline or phosphate buffer, carbonate buffer, Tris buffer, acetate buffer or borate buffer. Buffer, etc., or in a mixed solvent of these aqueous solvents and an organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, pyrrolidone,
-10 to +120 between the reactive liposome and the functional substance
℃, in the case of reaction with an amino group preferably 0 to 60 ℃,
More preferably 0 to 40 ° C., and in the case of reaction with a hydroxyl group or a thiol group, it is preferably carried out at 40 to 120 ° C. for 5 minutes to 1000 hours, preferably 30 minutes to 72 hours by a method of reacting with stirring, etc. Can be done easily. When immobilizing a protein such as an antibody or an antigen, it is -10 to + 100 ° C in an aqueous solvent having a pH of 4 to 12, preferably 6 to 10, and preferably 0 to 60 ° C in the case of a reaction with an amino group. , More preferably 0-40
In the case of a reaction with a hydroxyl group or a thiol group at a temperature of 50 ° C., the reaction is preferably performed at 50 to 80 ° C. for 30 minutes to 200 hours, preferably 1 minute to 48 hours with stirring. Outside of the above conditions, the stability of the liposome becomes poor, which is not preferable.

The immobilization reaction of a functional substance having an amino group, a hydroxyl group or a thiol group on the surface of a reactive liposome is schematically shown by the following formulas (3a) to (3c). In the formula, R and n are the same as described above.

Embedded image

After completion of the immobilization reaction, purification can be carried out, if necessary, by a method such as gel filtration, ultrafiltration, dialysis, centrifugation, stationary sedimentation separation and the like.

In the reactive liposome of the present invention, various substances can be encapsulated in the interior of the liposome, that is, in the inner aqueous phase of the liposome or in the membrane, by a known method, similar to general liposomes. Examples of the substance to be encapsulated include labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds, chemiluminescent compounds, and electrode sensitive compounds; external stimulus responses such as photoresponsive compounds, pH responsive compounds, and thermoresponsive compounds. Compounds; enzymes, antibodies, other proteins,
Physiologically active substances such as sugars, lipids, glycoproteins, glycolipids and hormones; drugs such as medicines and agricultural chemicals; water-soluble polymers such as polyacrylamide, polyvinyl alcohol, α-hydro-ω-hydroxy-polyoxyethylene and hyaluronic acid Kind and the like.

The encapsulation of the substance to be encapsulated in the aqueous phase in the liposome is carried out by using an aqueous solution in which the drug is dissolved in advance during the preparation of the reactive liposome, or after the preparation of the reactive liposome or the functional substance-immobilized liposome, the pH gradient is applied. Can be easily carried out by a method such as a method of encapsulating using a method or an osmotic pressure gradient method. When the substance to be encapsulated has a film-forming ability, when the substance is used together with a film-forming component to form a liposome during liposome formation,
The substance to be encapsulated is incorporated and encapsulated in the liposome membrane. After completion of the encapsulation operation, purification can be carried out, if necessary, by a method such as gel filtration, ultrafiltration, dialysis, centrifugation, stationary sedimentation separation or the like.

In the reactive liposome of the present invention, since the reactive cholesterol derivative represented by the general formula (1) has a sterol skeleton, this reactive cholesterol derivative is different from a linear polyoxyalkylene derivative or the like. In comparison, it is less likely to be detached from the liposome membrane. Therefore, the reactive liposome of the present invention has excellent long-term storage stability. Further, the reactive liposome of the present invention is (poly)
Since an oxyalkylene chain has been introduced, the effects of conventional (poly) oxyalkylene chain introduction, such as maintaining blood concentration for a long period of time, non-immunogenicity, and preventing leakage of substances encapsulated inside liposomes Can be expected.
Further, since the functional substance-immobilized liposome in which the functional substance is immobilized on the reactive liposome of the present invention, the functional substance is immobilized on the tip of the (poly) oxyalkylene chain,
The action of the functional substance is sufficiently exerted without being disturbed by the (poly) oxyalkylene chain.

The reactive liposome of the present invention has various functionalities such as drug carrier, liposome preparation, test agent, diagnostic agent, sensor, immobilized catalyst, bioreactor, bioelectronic device, microcapsule substitute, and cosmetic raw material. It can be used as a liposome or a carrier thereof.

In the production of the reactive liposome of the present invention, a polymerizable phospholipid is added as the other film-forming component together with the reactive cholesterol derivative represented by the general formula (1) to give a polymerizable reaction. Can be a liposome. As the polymerizable phospholipid, a known polymerizable phospholipid can be used.
-Di (2,4-octadecadienoyl) -3-phosphatidylcholine, as well as Shonoichi Nojima, Junzo Sunamoto, Keizo Inoue, "Liposome" p313, published by Minamiedo in 1988.
315 and the like. Among these, 1,2-di (2,4-octadecadienoyl) -3
-Phosphatidylcholine is preferred.

Polymerizable liposomes are prepared by subjecting them to UV, γ in the presence or absence of a photopolymerization initiator after liposome preparation.
Polymerization can be easily carried out by irradiation with light such as rays or electron beams, or by heating with a redox initiator system or in the presence of an azo initiator or an organic peroxide. Since the liposomes thus obtained after polymerization have excellent stability, they can be stably used as they are while being dispersed in an aqueous solution or prepared into powder by freeze-drying or the like. .

Next, the reactive endoplasmic reticulum other than the reactive liposome will be described. The reactive fat emulsion as a reactive endoplasmic reticulum comprises a reactive cholesterol derivative represented by the general formula (1), a vegetable oil such as soybean oil and safflower oil, and a phospholipid such as soybean lecithin and egg yolk lecithin, if necessary. Other additives to be added, for example, Intralipid (trademark of Otsuka Pharmaceutical Co., Ltd.), emulsification aid, stabilizer, isotonic agent,
An oil / fat mixture containing a fat-soluble drug such as a fat-soluble drug and a fat-soluble physiologically active substance is an emulsion emulsified in a water phase. In this reactive fat emulsion, the reactive cholesterol derivative represented by the general formula (1) and other vesicle-forming components are collected at the interface between the emulsified and dispersed oil droplets and the aqueous phase to form a reactive fat emulsion. The content of the reactive cholesterol derivative represented by the general formula (1) in the oil / fat mixture is 0.01 to 50 mol%, preferably 0.5 to
It is preferably 30 mol%.

The reactive fat emulsion of the present invention can be produced by a known method. For example, the reactive cholesterol derivative represented by the general formula (1), vegetable oil, phospholipid, and additives to be blended if necessary are mixed and heated, water is added to coarsely emulsify with a homomixer, and then Manton-Gaulin type It can be produced by a method such as homogenizing with a pressure injection type homogenizer. At this time, a reactive fat emulsion containing the drug in the fat emulsion can be obtained by mixing the fat-soluble drug into the oil / fat mixture as a raw material for production.

In the reactive fat emulsion thus obtained, the same functional substance can be easily immobilized as in the case of the reactive liposome. The reactive fat emulsion of the present invention, like the case of the reactive liposome,
It can be used as a drug carrier such as a drug, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, and a raw material for cosmetics.

Reactive micelles other than the above, which are reactive endoplasmic reticulum, are micelles containing the reactive cholesterol derivative represented by the general formula (1), and consist only of the reactive cholesterol derivative represented by the general formula (1). Even micelles, lecithin, other phospholipids, cholesterol,
It may be a micelle containing a fat-soluble drug such as a fat-soluble drug or other micelle component such as a fat-soluble physiologically active substance.
The reactive micelle can be formed by adding the reactive cholesterol derivative represented by the general formula (1) alone or a mixture of this and other micelle forming components to the aqueous phase at a concentration higher than the micelle forming concentration. it can. By mixing the fat-soluble drug in the manufacturing raw material, a reactive micelle containing the drug in the micelle can be obtained. The reactive micelle of the present invention can also immobilize a functional substance in the same manner as the reactive liposome. The reactive micelle of the present invention can also be used as a drug carrier such as a drug, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, and a raw material for cosmetics, as in the case of the reactive liposome.

[0042]

The reactive vesicle-forming agent of the present invention contains the reactive cholesterol derivative represented by the general formula (1). Therefore, the reactive vesicle containing the derivative has a (poly) oxyalkylene chain. In addition to being introduced, various functional substances can be easily and efficiently immobilized on the tip of the (poly) oxyalkylene chain by covalent bonding, and the amount of introduced functional substance can be increased. In this case, the reactive endoplasmic reticulum has excellent long-term storage stability.

Since the reactive endoplasmic reticulum of the present invention contains the reactive cholesterol derivative represented by the general formula (1) as an endoplasmic reticulum-forming component, the (poly) oxyalkylene chain is introduced and the endoplasmic reticulum is incorporated. In addition, various functional substances can be easily and efficiently immobilized by a covalent bond via a spacer composed of (poly) oxyalkylene, and the amount of the introduced functional substance can be increased. Moreover, the reactive endoplasmic reticulum of the present invention has excellent long-term storage stability.

[0044]

EXAMPLES The present invention will now be described in more detail by way of examples, but the present invention is not limited thereto. Synthesis Example 1 Toluene 500 ml, cholesterol 10 g (26 mmol) and sodium metal 0.6 g in an autoclave
(26 mmol) was added and the mixture was stirred for 30 minutes, 52 g (1.2 mol) of ethylene oxide was added, and the mixture was stirred at 100 ° C. for 24 hours. After completion of the reaction, the product was washed with 5% hydrochloric acid and distilled water, and then toluene was distilled off under reduced pressure to give an intermediate in which ethylene oxide was added to the hydroxyl group of cholesterol (hereinafter, referred to as Chol-PEG, a molecular weight of about 240).
0, and the number of moles of ethylene oxide added was about 46) (yield 100%). Furthermore, 5 g (2 mmol) of Chol-PEG and 0.32 g (2 mmol) of N, N'-carbonyldiimidazole were added to 10 ml of chloroform,
Stirred at room temperature for 6 hours. After completion of the reaction, the product was purified by reprecipitation in diethyl ether to obtain a white powdery reactive cholesterol derivative represented by the following formula (4). The progress of the reaction is
Chol-PEG in IR spectrum (KBr method)
Of hydroxyl group (OH stretching, 3430 cm ⁻¹ ) in the inside and formation of —OC (═O) N = bond (C═O stretching, 1760)
cm ^-1 ).

[0045]

[Chemical 6]

Example 1-1 Egg yolk phosphatidylcholine 20 mg (26 μmol), cholesterol 3.9 mg (10 μmol) and synthesis example 1
2.4 mg of the reactive cholesterol derivative obtained in (2) (10 wt% with respect to the above two) was placed in an eggplant-shaped flask,
After dissolving with 2 ml of benzene, freeze-drying was performed.
To this, 1 ml of physiological saline was added, and multi-layer liposomes were obtained by bath-type ultrasonic irradiation and a vortex mixer. Furthermore, with Extruder 3.0, 1.0,
A 0.2 μm polycarbonate membrane was sequentially passed through to obtain large unilamellar reactive liposomes. The particle size of the obtained reactive liposome was measured by a laser scattering particle size distribution meter (N
The particle size was 181 nm (CV value 15) as measured by using NICOMP370HPL (trademark) manufactured by ICOMP.
%)Met. After the above liposomes were allowed to stand at 5 ° C. for 3 weeks, the particle size was measured to be 195 nm (CV value 23
%) And was excellent in stability.

Example 1-2 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (5) was used as a reactive vesicle forming agent in an amount of 5 wt% with respect to the other two components. Reactive liposomes were obtained by using (particle size 185 nm, CV value 16%). Also 5 ℃
After standing for 3 weeks, the particle size is 199 nm and the CV value is 25.
%, And was excellent in stability.

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Example 1-3 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (6) was used as a reactive vesicle forming agent in an amount of 20 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 189 nm, CV value 18%). Also 5
The particle size was 194 nm and the CV value was 2 after standing at ℃ for 3 weeks.
It was 0% and was excellent in stability.

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Example 1-4 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (7) was used as a reactive vesicle forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 185 nm, CV value 15%). Also 5
The particle size after standing at ℃ for 3 weeks is 189 nm, CV value is 1
It was 7% and was excellent in stability.

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Example 1-5 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (8) was used as a reactive vesicle forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using this (particle size 188 nm, CV value 14%). Also 5
The particle size was 191 nm and the CV value was 1 after standing at ℃ for 3 weeks.
It was 9% and was excellent in stability.

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Example 1-6 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (9) was used as a reactive vesicle forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 182 nm, CV value 16%). Also 5
After standing for 3 weeks at ℃, the particle size is 190nm, CV value is 1
It was 8% and was excellent in stability.

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Example 1-7 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (10) was used as a reactive vesicle-forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 181 nm, CV value 14%). The particle size after standing at 5 ° C. for 3 weeks was 189 nm, and the CV value was 16%, indicating excellent stability.

[Chemical 12]

Example 1-8 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (11) was used as a reactive vesicle-forming agent in an amount of 10 wt% with respect to the other two components. Reactive liposomes were obtained by using (particle size 183 nm, CV value 15%). The particle size after standing at 5 ° C. for 3 weeks was 190 nm, and the CV value was 18%, indicating excellent stability.

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Example 1-9 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (12) was used as a reactive vesicle forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 182 nm, CV value 15%). The particle size after standing at 5 ° C. for 3 weeks was 191 nm, and the CV value was 18%, indicating excellent stability.

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Example 1-10 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (13) was used as a reactive vesicle forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using this (particle size 180 nm, CV value 15%). Further, the particle size after standing at 5 ° C. for 3 weeks was 189 nm and the CV value was 17%, which was excellent in stability.

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Example 1-11 In the same manner as in Example 1-1, except that the reactive cholesterol derivative of the following formula (14) was used as a reactive vesicle-forming agent in an amount of 10 wt% with respect to the other two components. A reactive liposome was obtained by using the above (particle size 182 nm, CV value 16%). The particle size after standing at 5 ° C. for 3 weeks was 192 nm, and the CV value was 19%, indicating excellent stability.

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Examples 1-12 As in Example 1-1, but using 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine (DODP) instead of egg yolk phosphatidylcholine. Small unilamellar liposomes with a particle size of 254 nm (CV value 23%) were obtained. This was irradiated with 0.75 Mrad γ-rays to polymerize DODP. The obtained polymerized liposomes were subjected to gel filtration using Sephadex G-50,
Freeze-drying gave powdery samples. This liposome powder could be regenerated by swelling with physiological saline.

Example 2-1 1 μg / ml horseradish peroxidase (hereinafter abbreviated as HRP) /0.1 M was added to 500 μl of the liposome solution (solid content: about 0.25%) obtained in Example 1-1. Add 100 μl of phosphate buffer (pH 7.5) at 2 ℃ at 2 ℃
The HRP was immobilized on the liposome by stirring for 4 hours. This was subjected to gel filtration using Sephadex G-50, and the liposome-containing fraction was fractionated to obtain a HRP-immobilized liposome.

Examples 2-2 to 2-12 HRP-immobilized liposomes were obtained in the same manner as in Example 2-1 except that the reactive liposomes obtained in Examples 1-2 to 1-12 were used.

Reference Example 500 μl of the HRP-immobilized liposome solution obtained in Examples 2-1 to 2-12 was added with 1,2-HRP substrate.
100 μl of phenylenediamine solution (10 mmol / l) was added, and the mixture was incubated at 30 ° C. for 10 minutes, and further added to 0.1.
When 10 μl of 1N sulfuric acid was added, a brown coloration was observed in all.

Comparative Example Using only 20 mg (26 μmol) of egg yolk phosphatidylcholine and 3.9 mg (10 μmol) of cholesterol, a solid content of 2. was obtained in the same manner as in Example 1-1.
5% multilamellar liposomes were obtained. HRP was made to act on this in exactly the same manner as in Example 2-1, and purified by gel filtration. 500 μl of the obtained liposome solution was added with 1,2
-Phenylenediamine solution (10 mmol / l) 100 μl
Was added, the mixture was incubated at 30 ° C. for 10 minutes, and 10 μl of 0.1N sulfuric acid was further added, but no color development was observed.

From the results of Reference Examples and Comparative Examples, it was confirmed that the reactive liposomes containing the reactive cholesterol derivative as the membrane-forming component can easily immobilize HRP while retaining the activity of HRP.

Claims (2)

[Claims] 1. A reactive endoplasmic reticulum forming agent comprising a reactive cholesterol derivative represented by the following general formula (1). Embedded image [In the Formula, OA is a C2-C4 oxyalkylene group, n
Is the average number of moles of oxyalkylene groups added, which is from 1 to 100.
Represents a positive number of zero. When n is 2 or more, the oxyalkylene groups may be the same or different and may be added randomly or in a block. R
Represents a hydrogen atom or a methyl group, X represents a divalent organic residue, and p represents 0 or 1. ] 2. A reactive endoplasmic reticulum comprising one or more of the reactive endoplasmic reticulum forming agent according to claim 1 as an endoplasmic reticulum forming component.