JP3498336B2 - Phospholipid derivatives - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel and useful phospholipid derivative. More specifically, it is used for the production of various functional liposomes or vesicles such as fat emulsions, such as pharmaceutical carriers, test agents, diagnostic agents, sensors, immobilized catalysts, bioreactors, bioelectronic devices, and microcapsule substitutes. And phospholipid derivatives. 2. Description of the Related Art Liposomes are vesicles composed of a phospholipid bilayer membrane, and their application in various fields has been attempted. In particular, application to pharmaceutical carriers, sensors for diagnosis and detection, etc., has attracted attention.However, immobilizing a functional substance on the liposome surface or in a membrane to provide various functions, and reducing the blood concentration of liposome Maintaining is a major issue. Heretofore, regarding the immobilization of a functional substance on the surface of a liposome or in a membrane, the aminoethylcarbamylmethyl group substituted on a polysaccharide on the surface of a liposome coated with a pullulan derivative is attached to a γ-maleimidobutyloxysuccinate. A method in which an antibody fragment is bound via nimidyl (Biochem. Biophys. Acta., 898 , 323 (1987)), or glycolipids are added to liposome membrane-forming components in advance, and liposome formation is followed by periodate oxidation. And immobilization by reacting the resulting aldehyde group with the 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 the preparation of the liposome. There is a problem that by-products and impurities are mixed, and the liposome membrane is greatly damaged. [0005] On the other hand, it has been pointed out that when liposomes are administered into a living body, most of them are trapped by reticulo-organs such as liver and spleen, so that sufficient effects cannot be obtained (Cancer Res., 43 , 5328 (1983)). Therefore, as a method of improving the problem of being trapped by the reticulo-organs or the problem of low stability such as disintegration and aggregation of the liposome itself, a polyethylene glycol chain is introduced into the surface of the liposome. (For example, Japanese Patent Application Laid-Open No. 1-249717, FEBS letters,
268 , 235 (1990)). In addition, it has been shown that liposomes modified with polyethylene glycol can maintain blood concentrations for a long period of time (Biochem. Biophyl.
s. Acta., 1066 , 29-36 (1991)). However, since the liposome into which the polyethylene glycol chain obtained by such a method has been introduced does not react with the functional substance, the functional substance cannot be immobilized on the liposome surface. Further, JP-A-4-346918 discloses that a liposome having a maleimide group is first reacted with a protein having a thiol group (thiolated protein), and then a polyalkylene glycol having a thiol group added to the remaining maleimide group. It is described that by reacting a compound containing a (thiolated polyalkylene glycol) moiety, drug-containing antibody-bound liposomes with improved uptake in reticulo-organs can be obtained. But,
In this liposome, the antibody is hidden under the polyalkylene glycol layer, and the reaction with the antigen at the target site is hindered. Therefore, there is a problem that the expected effect cannot be sufficiently obtained. SUMMARY OF THE INVENTION An object of the present invention is to provide a novel and useful compound capable of simply and efficiently immobilizing various functional substances at the tip of a polyalkylene oxide chain by a covalent bond. It is to provide a phospholipid derivative. Means for Solving the Problems The present invention is a phospholipid derivative represented by the following general formula [1]. Embedded image [Wherein R ¹ C (= 0) and R ² C (= 0) have 3 carbon atoms
Represents an acyl residue of up to 30 fatty acids, which may be the same or different. R ³ is a hydrogen atom or a methyl group, AO an oxyalkylene group having 2 to 4 carbon atoms, n represents an average addition mole number of the oxyalkylene group, the positive number of 1 to 1,000. When n is 2 or more, the oxyalkylene groups may be the same or different, and may be added randomly or in blocks. q represents an integer of 0 to 3, and M represents a hydrogen atom or an alkali metal atom. In the general formula [1], R ¹ C (= 0), R ²
The acyl residue of the fatty acid represented by C (= 0) has 3 to 30 carbon atoms (including carbon atoms of a carbonyl group), preferably 8 carbon atoms.
~ 20 acyl residues. Specific examples of such acyl residues include propionic acid, butyric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. Acyl residues of saturated fatty acids such as acid, lignoceric acid, sorotic acid, montanic acid, melicic acid, 2-ethylhexanoic acid; oleic acid, linoleic acid, linolenic acid, erucic acid, 2,4-octadecadienoic acid, etc. acyl residue of branched fatty acids such as isostearic acid; acyl residue of an unsaturated fatty acid ricinoleate Lumpur acid, an acyl residue of a fatty acid having a hydroxyl group in the alkyl group, such as 12-hydroxystearic acid. When the phospholipid derivative of the present invention is used for producing a liposome or a fat emulsion, R ¹ C (=
0), the acyl residue of a fatty acid represented by R ² C (= 0) can be formed from myristic acid, palmitic acid, stearic acid, oleic acid, 2,4-octaic acid because a stable liposome or fat emulsion can be formed. Acyl residues of decadienoic acid are preferred, and acyl residues of palmitic acid, stearic acid and oleic acid are particularly preferred. R ¹ C (= 0) and R ²
C (= 0) may be the same or different. The oxyalkylene group represented by AO in the general formula [1] is an oxyalkylene group having 2 to 4 carbon atoms, such as an oxyethylene group, an oxypropylene group, an oxytrimethylene group, and 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 polymerization of an alkylene oxide such as 1-butene oxide, 2-butene oxide, and tetrahydrofuran. N in the general formula [1] is a positive number of 1 to 1000, preferably 10 to 300, and more preferably 20 to 120. When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks. When imparting hydrophilicity, AO is preferably one to which ethylene oxide is independently added, 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 polyalkylene oxide chain, the number of moles other than ethylene oxide is increased. R ^{3 in the} general formula [1] is a hydrogen atom or a methyl group. M in the general formula [1] is a hydrogen atom or an alkali metal atom such as sodium or potassium. In order to produce the phospholipid derivative represented by the general formula [1], for example, a carboxyl group is added at one end.
A polyalkylene glycol derivative having a hydroxyl group at the other end, and dicyclohexylcarbodiimide (DCC)
And reacted with phosphatidylethanolamines, and then the resulting compound was reacted with N,
It can be easily produced by a method of reacting N'-carbonyldiimidazole (CDI) or the like. The reaction formula when α-hydro-ω-carboxyl- polyoxyethylene is used as the polyalkylene glycol derivative and dipalmitoylphosphatidylethanolamine (DPPEA) or a salt thereof is used as the phosphatidylethanolamine is shown below. In the formula, M and n represent the same as described above. Embedded image In addition to the above-mentioned production method, a compound in which the carboxyl group of the above polyalkylene glycol derivative is converted to an acid chloride using thionyl chloride, isobutyl chloroformate, or the like, or activated by a succinimide, carbonyldiimidazole, or the like. An esterified compound;
It can also be easily produced by reacting phosphatidylethanolamines with CDI and reacting the resulting compound with CDI. These reactions can be carried out without solvent or with water, toluene, benzene, acetonitrile, methanol, 1,
In a solvent such as 4-dioxane, tetrahydrofuran or chloroform, -4 in the atmosphere or under an inert gas atmosphere such as nitrogen, argon, helium or carbon dioxide.
0 to 120 ° C, preferably 0 to 60 ° C for 10 minutes to 2
It is desirable to carry out by stirring for 40 hours, preferably for 1 hour to 48 hours. After completion of the reaction, the reaction mixture obtained is purified as it is or by distillation, recrystallization, reprecipitation, treatment with an adsorbent, column treatment, gel filtration, ultrafiltration, dialysis, etc.
After isolation, it can be used as an endoplasmic reticulum-forming component and the like. Here, the endoplasmic reticulum means particles having a structure in which a hydrophilic group of an endoplasmic reticulum-forming component is oriented toward an aqueous phase at an interface. Specific examples include liposomes, which are closed vesicles composed of a bilayer membrane, fat emulsions in which a mixture of vegetable oil, phospholipid, and the like are emulsified, micelles, and the like. The phospholipid derivative of the present invention has a functional group such as an amino group, a hydroxyl group or a thiol group, particularly an oxycarbonylimidazole group which is highly reactive with a primary amino group. Easily reacts with a functional substance having an appropriate functional group to form a covalent bond. Therefore, by using a phospholipid derivative of the present invention as vesicle forming components, it is possible to introduce a poly A alkylene oxide chain in the endoplasmic reticulum, it is possible to impart reactivity to a functional substance having the functional group , Reactive vesicles can be produced. When the endoplasmic reticulum is produced using the phospholipid derivative of the present invention, the phospholipid derivative may be used alone or in combination of two or more, or other vesicle-forming components capable of forming an endoplasmic reticulum, such as soybean Lecithin, yolk lecithin, that
Other phospholipids, cholesterol and Intralipid (Otsuka Pharmaceutical Co., trademark), soybean oil, and mixed with such safflower oil can be used, initially reactive fatty emulsion reactive liposome, reaction Reactive vesicles such as sexual micelles. In this case, the endoplasmic reticulum can be produced by a known method. The reactive endoplasmic reticulum obtained using the phospholipid derivative of the present invention is capable of covalently bonding various functional substances using the oxycarbonylimidazole group in the compound represented by the general formula [1] as a functional group. Can be introduced. Next, each reactive vesicle produced using the phospholipid derivative of the present invention will be described in detail.
Reactive liposomes, which are typical reactive vesicles, contain a phospholipid derivative as a membrane-forming component (vesicle-forming component). The content of the phospholipid derivative is 0.01% based on the total amount of the phospholipid derivative and other film forming components.
５０50 mol%, preferably 0.5-30 mol%. If the amount is less than 0.01 mol%, the expected effect is small, and if it exceeds 50 mol%, the stability of the liposome is reduced, so that it is not generally used. The phospholipid derivatives can be used alone or in combination of two or more. As the other membrane-forming components used in admixture with the phospholipid derivative, those conventionally used as liposome membrane-forming components can be used without limitation. Specifically, diphosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, soy lecithin,
Egg yolk lecithin, phosphatidylcholine , phosphatidyl
Phospholipids such as glycerol and polymerizable phospholipids having an unsaturated group in the fatty acid moiety; glycolipids such as sulfoxyribosyl diglyceride, digalactosyl diglyceride, and lactosyl diglyceride; nonpolar lipids such as cholesterol; Surfactant, phosphatidyl polyethylene glycol, "Biochem. Biophys. Acta., 106
6 , 29-36 (1991) ", a reaction product of phosphatidylethanolamine with polyethylene glycol, a mixture thereof and the like. The reactive liposomes, phospholipid derivatives and lecithin, other phospholipids, other film-forming components, such as cholesterol, are dissolved in a suitable solvent such as an organic solvent, ex True John method, vortex mixer method Liposomes by various known methods such as ultrasonic method, surfactant removal method, reverse layer evaporation method, ethanol injection method, prevesicle method, French press method, W / O / W emulsion method, annealing method, freeze-thaw method, etc. Can be produced by In addition, by selecting these production methods, multilamellar liposomes, small unilamellar liposomes,
Reactive liposomes having various sizes and shapes, such as large unilamellar liposomes, can be produced. The reactive liposome thus obtained has an oxycarbonylimidazole group bonded to the inner and outer surfaces of the liposome membrane via a polyalkylene oxide spacer, so that the reactive liposome has an amino group, a hydroxyl group, a thiol group and the like. A functional substance having a functional group, particularly a primary amino group, is efficiently and easily chemically and chemically bonded to a liposome bilayer membrane by a urethane bond, a carbonate bond or a thiocarbonate bond via a spacer made of polyalkylene oxide. Can be immobilized. The functional substance that can be immobilized on the reactive liposome includes, for example, a dye, a dye, a radiolabeled compound,
Fluorescent compounds, chemiluminescent compounds, labeled substances, such as the electrode sensitive compound; photoresponsive compounds, pH responsive compound, an external stimulus responsive compound such as thermally responsive compound; enzymes, antibodies, other proteins, sugars, lipids , Glycoproteins, glycolipids, hormones and other physiologically active substances; The immobilization reaction of the functional substance on the reactive liposome can be carried out by using an aqueous solvent such as various buffers, or an aqueous solvent such as acetone, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, or dimethylamine. In a mixed solvent with an organic solvent such as cetamide, dimethyl sulfoxide, and pyrrolidone, the reactive liposome and the functional substance are used at -10 to 120 ° C, preferably 0 to 60 ° C, and more preferably in the case of a reaction with an amino group. Is 0-40
C., in the case of a reaction with a hydroxyl group or a thiol group, it can be easily carried out in one step, for example, by reacting at 40 to 120 ° C. with stirring for 5 minutes to 1000 hours, preferably 30 minutes to 72 hours. it can. Outside these conditions, the stability of the liposome deteriorates, which is not preferable. The reaction of immobilizing a functional substance having an amino group on the surface of a liposome is schematically shown as follows. In the formula, R ³ , AO, and n represent the same as described above. Embedded image Various substances can be encapsulated in the reactive liposome by a known method as in the case of general liposomes. Examples of the substance to be encapsulated include labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds, and chemiluminescent compounds; and external stimulus responses such as photoresponsive compounds, pH-responsive compounds, thermoresponsive compounds, and electrode-sensitive compounds. Sex compounds; enzymes, antibodies, other proteins, sugars,
Physiologically active substances such as lipids, glycoproteins, glycolipids, and hormones; pharmaceuticals; and water-soluble polymers such as polyacrylamide, polyvinyl alcohol, polyethylene glycol, and hyaluronic acid. After completion of the immobilization reaction or the encapsulation operation, if necessary, purification can be performed by a method such as gel filtration, ultrafiltration, dialysis, centrifugation, and sedimentation by standing. The liposomes immobilized functional material is a polyalkylene oxide tip functional material strand is immobilized Tei Runode, the action of the functional substance without being disturbed by the polyalkylene oxide chain is fully exhibited Is done. Also,
Since the polyalkylene oxide chain has been introduced, the effects of the conventional polyalkylene oxide chain introduction, such as maintenance of blood concentration over a long period of time, non-immunogenicity, and effects of preventing leakage of substances encapsulated inside the liposome are also exhibited. Can be expected. For this reason, the reactive liposome can be used as various functional liposomes such as a drug carrier, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, a bioreactor, a bioelectronic element, and a microcapsule substitute. In the production of the reactive liposome, a polymerizable reactive liposome can be obtained by blending a polymerizable phospholipid as another membrane-forming component. Known polymerizable phospholipids can be used as the polymerizable phospholipid. For example, in addition to 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine,
Edited by Shojima Nojima, Junzo Sunamoto and Keizo Inoue, liposomes published by Nankodo in 1988, pp. 313-315, and the like. Among these, 1,2-di (2,4
-Octadecadienoyl) -3-phosphatidylcholine is preferred. After preparing the liposome, the polymerizable liposome can be treated with UV, γ in the presence or absence of a photopolymerization initiator.
Polymerization can be easily carried out by irradiation with light such as a beam or an electron beam, or by heating with a redox initiator system or in the presence of an azo-based initiator or an organic peroxide. Since the liposome after polymerization obtained in this manner has excellent stability, it can be stably used while being dispersed in an aqueous solution or prepared in a powder form by freeze-drying or the like. . The reactive lipid emulsion as another reactive endoplasmic reticulum is added with the phospholipid derivative of the present invention, 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, for example, Intralipid (trademark, manufactured by Otsuka Pharmaceutical Co., Ltd.), an emulsifier,
An oil or fat mixture containing a stabilizer, an isotonic agent, a fat-soluble drug, a fat-soluble physiologically active substance and the like is emulsified. The content of the phospholipid derivative in the fat mixture is 0.01 to
It is desirably 50 mol%, preferably 0.5 to 30 mol%. The reactive fat emulsion can be produced by a known method. For example, phospholipid derivatives, vegetable oils,
Mixing and heating a phospholipid and additives to be blended if necessary, adding water, coarsely emulsifying with a homomixer or the like, and then homogenizing with a Manton-Gaurin type pressure injection homogenizer or the like. Can be. In the reactive fat emulsion thus obtained, the same functional substance can be easily immobilized in the same manner as in the case of the reactive liposome. Therefore, the reactive fat emulsion can be used as a pharmaceutical carrier, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, and the like. [0035] Reactive micelle is a reactive vesicle other than the above, be comprised of only the phospholipid derivative of the present invention, lecithin, other phospholipids, other of cholesterol etc. <br/> A component may be contained. The reactive micelle can immobilize the functional substance in the same manner as the reactive fat emulsion, and can be used for the same purpose. The phospholipid derivative of the present invention is novel and useful. By using the phospholipid derivative of the present invention as an endoplasmic reticulum-forming component, a polyalkylene oxide chain can be introduced into the endoplasmic reticulum, and various functional substances can be easily and efficiently covalently bonded to the tip of the polyalkylene oxide chain. Thus, the amount of the functional substance introduced can be increased. EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 α-hydro-ω-carboxyl -polyoxyethylene
( Mw: about 3000, degree of polymerization: about 65) 1.0 g (0.
3 mmol) and 68 mg (0.3 mmol) of dicyclohexylcarbodiimide were dissolved in 10 ml of ethyl acetate and stirred at 5 ° C. for 1 hour. Next, 217 mg of dipalmitoyl phosphatidylethanolamine (0.3 mm
ol) was dissolved in 10 ml of an ethyl acetate solution, and the mixture was further stirred for 6 hours, allowed to stand at 0 ° C. overnight, and the precipitate was removed by filtration. Next, carbonyldiimidazole 4 was added to the filtrate.
9 mg (0.3 mmol) was added, the mixture was stirred at room temperature for 1 hour, and the obtained reaction mixture was diluted with 100 ml of diethyl ether.
The precipitate was collected by filtration, and the following phospholipid derivative was obtained in the form of a white powder (yield: 88%). The progress of the reaction was determined by IR spectrum (KBr method). As an intermediate, disappearance of the amino group (N ⁺ H ₂ stretch, 3000 cm ^-1 ) in the phosphatidylethanolamine and amide bond (C = O
Stretch, 1647 cm ^-1 ), and the target product was the disappearance of the hydroxyl group (OH stretch, 3428 cm ^-1 ) at the terminal of the polyethylene glycol derivative and oxycarbonylylation.
Formation of midazole bond (carbonyl bond) (1760c
m ^-1; C = a more confirmed in O Shin contraction). Embedded image The results of NMR and IR of the above phospholipid derivative are as follows. ¹ H-NMR (CDCl ₃ , δ; ppm, 270 MHz) 8.14 (a; s, 1H) 7.43 (b; t, 1H, J = 1.3) 7.08 (c; t, 1H, J = 0.8) 5.23 (6; m, 1H) 4.67 (d; t, 2H, J = 2.5) 4.29 (8; m, 2H) 4.00 (5, 7; m, 4H) 3.64 (e; m, about 240H) 3.06 (f; t, 2H, J = 7.3) 2.31 (4, 4 '; m, 4H) 1.60 (3, 3 '; m, 4H) 1.26 (2, 2'; m, 48H) 0.88 (1, 1 '; t, 6H, J = 6.4) IR; (KBr, cm ^-1 ) 1760 ( Oxycarbonylimidazole bond , C = O
Stretching vibration) 1728 (ester, C = O stretching vibration) 1647 (amide, C = O stretching vibration) 1526 ( amide , NH bending vibration) 1465 (C-H bending vibration) Example 2 Example 1 The following phospholipid derivatives were obtained in the same manner as described above, except that α-carboxymethyl-ω-hydroxy -polyoxyethylene ( degree of polymerization: about 35) was used as the polyalkylene glycol derivative. Embedded image The results of NMR and IR of the above phospholipid derivative are as follows. ¹ H-NMR (CDCl ₃ , δ; ppm, 270 MHz) 8.14 (a; s, 1H) 7.43 (b; t, 1H, J = 1.3) 7.08 (c; t, 1H, J = 0.8) 5.23 (6; m, 1H) 4.67 (d; t, 2H, J = 2.5) 4.29 (8; m, 2H) 4.00 (5, 7; m, 4H) 3.64 (e; m, about 240H) 2.31 (4, 4 '; m, 4H) 1.60 (3, 3'; m, 4H) 1.26 (2, 2 '; m, 48H) 0.88 (1, 1 '; t, 6H, J = 6.4) IR; (KBr, cm ^-1 ) 1760 ( oxycarbonylimidazole bond , C = O
Stretching vibration) 1728 (ester, C = O stretching vibration) 1647 (amide, C = O stretching vibration) 1526 ( amide , NH bending vibration) 1465 (C-H bending vibration) Example 3 Example 1 As above, except that the polyalkylene glycol derivative is Was used to obtain the following phospholipid derivative. Embedded image The results of NMR and IR of the above phospholipid derivative are as follows. ¹ H-NMR (CDCl ₃ , δ; ppm, 270 MHz) 8.14 (a; s, 1H) 7.43 (b; t, 1H, J = 1.3) 7.08 (c; t, 1H, J = 0.8) 5.23 (6; m, 1H) 4.67 (d; t, 2H, J = 2.5) 4.29 (8; m, 2H) 4.00 (5, 7; m, 4H) 3.60 (e, f, g; m, about 95H) 3.06 (j; t, 2H, J = 7.3) 2.31 (4, 4 '; m, 4H) 60 (3, 3 '; m, 4H) 1.26 (2, 2'; m, 48H) 1.12 (h; m, 15H) 0.88 (1, 1 '; t, 6H, J = 6) .4) IR; (KBr, cm ^-1 ) 1760 ( oxycarbonylimidazole bond , C = O
Stretching vibration) 1728 (ester, C = O stretching vibration) 1647 (amide, C = O stretching vibration) 1526 ( amide , NH bending vibration) 1465 (CH bending vibration)

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Claims (1)

(57) [Claim 1] A phospholipid derivative represented by the following general formula [1]. Embedded image [Wherein R ¹ C (= 0) and R ² C (= 0) have 3 carbon atoms
Represents an acyl residue of up to 30 fatty acids, which may be the same or different. R ³ is a hydrogen atom or a methyl group; AO is an oxyalkylene group having 2 to 4 carbon atoms; n is an average addition mole number of the oxyalkylene group;
Represents a positive number of 00. When n is 2 or more, the oxyalkylene groups may be the same or different, and may be added randomly or in blocks.
q represents an integer of 0 to 3, and M represents a hydrogen atom or an alkali metal atom. ]