JPH0523576A - Liposome vesicle - Google Patents

Abstract

PURPOSE:To provide a fluidity quite close to a viable tissue membrane by using phospholipid expressed by formula (I) as a membraneous lipid ingredient. CONSTITUTION:Phospholipid expressed by formula (I) (R is a 10 to 25C acyl group of a chained terpene structure with a plurality of methyl groups; X is a hydrophilic group, yet it referred to here is a hydrophilic group required for a chemical compound expressed by formula (I) to assume a molecular aggregate state such as libosome or micelle. An ionio group which is present in the molecule partly represents any appropriate pair of ion and salt) is used as a membraneous lipid ingredient. That is, a phospholipid where a hydrophobic part of a chained terpene structure having methyl groups is linked to a glycerol part using ester instead of ether as known conventionally as a linkage technique. Thus an isoprenoid phospholipid which can be synthesized easily and chemically stable is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a liposome having fluidity close to that of a biological membrane, chemically stable and excellent in biocompatibility, and a method for producing the liposome.

[0002]

2. Description of the Related Art Generally, it is known that when an amphipathic molecule such as a phospholipid is dispersed in water, a particular molecular aggregate state is obtained. Among them, the liposome is a closed endoplasmic reticulum formed of a lipid bilayer membrane and has an aqueous layer inside thereof, so that in recent years, in the fields of medicine and pharmacy, the liposome has been made to retain a water-soluble substance so as to be used as a drug carrier or diagnostic. Many attempts have been made to use it as a medicine (for example,
Sunamoto et al., Bioscience and Industry, Volume 47,
475 pages, 1989). In addition, the use of liposomes in cosmetics and the like has been attempted, which utilizes the water-retaining and moisturizing effects of liposomes.

As described above, the most important factor in utilizing phospholipids in water as liposomes or emulsions is whether they can be easily dispersed to obtain a uniform dispersion.
In addition, it is whether or not the obtained dispersion is stable. Therefore, it goes without saying that it is extremely important to use a material having good dispersibility and stability. For example, taking a liposome as an example, the fluidity of the liposome lipid bilayer membrane largely changes with temperature (gel-liquid crystal phase transition). It is known that the mobility of molecules in the bilayer film in the gel state and the liquid crystal state is much larger in the liquid crystal state in both lateral diffusion, flip-flop, and exchange. Generally, hydrophobic fatty acid residues having a small number of carbon atoms and liposomes composed of lipids with a high degree of unsaturation have high membrane fluidity, while those that are saturated and have a relatively large number of carbon atoms have low membrane fluidity, The phase transition temperature is also high. Therefore, by changing the chain length and the degree of unsaturation of the fatty acid residue of the lipid used, it is possible to control the membrane fluidity of the liposome and the lipid dispersibility and membrane barrier ability closely related thereto.

For example, a lipid containing an unsaturated fatty acid as a constituent, such as egg yolk phosphatidylcholine, is in a liquid crystal state at room temperature or higher because it has a low phase transition temperature and forms a soft film. Lipids with unsaturated fatty acids are indispensable to the living body because the biological membrane in the liquid crystal state acts as a barrier, and the fact that biological membranes acquire such properties is due to the rapid change of environmental factors such as temperature. On the other hand, it is considered that there was an advantage that the physical properties of the film changed due to buffering. Such a phenomenon is also important when using liposomes as a drug carrier. For example, when incorporating a hydrophobic drug into a liposome membrane, egg yolk phosphatidylcholine containing unsaturated fatty acids is more preferable than liposomes consisting only of saturated fatty acids. In many cases, the dispersibility is good and the encapsulation efficiency is high.

However, the polyunsaturated fatty acid contained in egg yolk phosphatidylcholine is susceptible to a peroxidation reaction by oxygen, and its storage stability is a major drawback. Therefore, from the viewpoint of stability, it is advantageous to use a phospholipid consisting only of saturated fatty acids that are not easily attacked by oxygen.
However, for example, even when a liposome is prepared using dimyristoylphosphatidylcholine, which is a naturally occurring saturated phospholipid, as a membrane component, it is extremely difficult to retain a water-soluble substance such as glucose in the liposome above the phase transition temperature. Further, it is known that even if a liposome composed only of dipalmitoylphosphatidylcholine is prepared, it is unstable, and that it immediately aggregates and precipitates. In general, phospholipids containing only saturated fatty acids have a dense arrangement (so-called hard but brittle film) below the phase transition temperature, which is inflexible and tends to eliminate foreign molecules and cause phase separation. It is impractical to prepare liposomes.

Therefore, when liposomes are used in the fields of biochemistry and pharmaceuticals, it is known that they are chemically unstable in order to obtain a fluid membrane close to a biological membrane, and naturally-occurring unsaturated fatty acids. The current situation is to use mixed lipids containing sucrose, or to mix cholesterol with lipids having a high phase transition temperature. Such excellent dispersibility, good film fluidity, and excellent chemical stability are fundamentally contradictory properties. Phosphorus composed only of saturated fatty acids, which have been conventionally used, is used. Whether lipids or phospholipids with unsaturated fatty acids,
No material could satisfy all of these requirements alone.

[0007] Therefore, as a result of an examination to search for a material which can satisfy all of these properties, the material was sought for a biological membrane of bacteria. Unlike animals and plants, bacteria usually do not contain polyunsaturated fatty acids in biological membranes. It has been 30 years since the first discovery of branched fatty acids (isoacids and antiisoacids) as the major fatty acids in bacterial lipids (S.
Akashi and K. Saito, J. Biochem., 47, 222, 19
60 years). At present, more than several hundred types of bacteria that have branched fatty acids as major fatty acids of biological lipids are known (T. Kaned
a, Bacteriol. Rev., 41, 391, 1977).

Then, using this as a model, diacylphosphatidylcholine having various branched fatty acids was recently synthesized, and the phase transition temperature was measured. As a result, it was clarified that the lipid having branched fatty acid had a lower phase transition temperature than the phospholipid having linear acid, and the difference was about 16 to 28 ℃. In other words, branched fatty acids contribute to improving the fluidity of bacterial biomembranes than the corresponding straight-chain acids (Satoshi Kaneda, Bioscience and Industry, 48, 229).
Page, 1990). Unlike the polyunsaturated fatty acids, these branched fatty acids are unlikely to be attacked by oxygen and are chemically stable, and are considered to be desirable materials. However, iso-acids and anti-iso-acids do not exist universally in nature, and since these are usually mixtures with different hydrophobic structure, separation and purification are very difficult. The remaining means is chemical synthesis, but the readily available raw materials are limited, and it is considered to be a major drawback that it is not suitable for large-scale synthesis because the number of steps for carbon chain extension from there is too many.

In order to solve such a problem, biomembranes of archaebacteria have been attracting attention in recent years. Archaea in 1977 in Woes
This is a concept proposed by e et al. by comparing the nucleotide sequences of 16S rRNAs of many organisms, and three groups of highly halophilic bacteria, sulfur-dependent highly thermophilic bacteria, and methanogenic bacteria are currently known (compounds). As a book, Yosuke Koga, Archaea, University of Tokyo Press, 1988). The polar lipids of archaebacteria are all glycerolipids having an ether bond as far as known so far, and their main feature is that the hydrocarbon chain is a saturated isoprenoid having 20 or 40 carbon atoms. Since saturated isoprenoids are also less susceptible to oxygen attack and are chemically stable, it is expected that materials with unique properties can be obtained by designing and synthesizing artificial lipids using such lipids as models.

Since chain isoprenoids are relatively easily available as compared with isoacids and antiisoacids, studies on lipids incorporating these in the hydrophobic part have recently been reported (K. Yamauchi). et al, Biochim. Biophys. Acta
1003, 151 pages, 1989, K. Yamauchi et al, J. Am. Ch
em. Soc., 112, 3188, 1990, LC Stewartet a
l, Chem. Phys. Lipids 54, 115, 1990, Yamauchi et al., Proceedings of Spring Meeting of the Chemical Society of Japan, 1990, 1793.
Pp. 1794, Toda et al., Proceedings of the Annual Meeting of the Chemical Society of Japan, 1990, p. 1793, RA Moss et al., Tetrahedron
Lett., Vol. 31, p. 7559, 1990, JP-A-2-288849). As a result, it was found that the lipid bilayer membrane formed from the isoprenoid-type lipid has a low phase transition temperature and a high membrane barrier ability. However, in the molecular design of isoprenoid-type artificial glycerolipids that have been reported so far, since the method of connecting the glycerol part and the hydrocarbon chain is the same ether type as the biological membrane of archaea, the synthetic method is poor in versatility and large-scale The major drawback was that it was not suitable for synthesis.

[0011]

As described above, there has been no material that satisfies all of our requirements, such as the phospholipids that are conventionally used only of saturated fatty acids and the phospholipids having unsaturated fatty acids. In particular, branched fatty acids such as isoacids and antiisoacids and lipids having a chain isoprenoid skeleton are expected to have promising properties, but these are extremely difficult to isolate and purify from nature and to chemically synthesize. Met.

Therefore, the object of the present invention is a property that could not be achieved by the phospholipids which are composed only of saturated fatty acids and the phospholipids having unsaturated fatty acids, which have been conventionally used, specifically, a soft film having a phase transition point of room temperature or lower Is to provide isoprenoid-type phospholipids that are chemically stable, and to provide liposome vesicles having a fluidity extremely close to that of a biological membrane using the isoprenoid-type phospholipid.

[0013]

The above-mentioned problems are solved by the following general formula (I)
It was achieved by a liposome vesicle characterized by using a phospholipid represented by as a membrane lipid component. General formula (I)

[0014]

[Chemical 2]

That is, in the present invention, a method for linking a hydrophobic part having a chain terpene structure having a plurality of methyl groups and a glycerol part is a phospholipid in which an ester bond is used instead of the conventionally known ether bond, and a liposome membrane lipid component is used. It is characterized by being used as.

In the formula, R has 10 to 25 carbon atoms and represents an acyl group having a chain terpene structure having a plurality of methyl groups. The acyl group having a chain terpene structure in the present invention means an acyl group whose structure is the same as or similar to a terpene having a chain structure, and which is derived from a natural chain terpene or a synthetic terpene. Is included. The acyl group of this chain terpene structure usually has a carbon number of 10 to 25 and has a plurality of branched methyl groups, and these are appropriately functionalized from phytol and isophytol which are diterpenes, and farnesol which is a sesquiterpene. It is possible to induce by group conversion. It is important for the present invention that the double bond existing in these terpenes is reduced to a saturated bond. Examples of such an acyl group are 3,7,11-trimethyldodecanoyl group and 3,7,11,15-
Examples thereof include a tetramethylhexadecanoyl group and a 5,9,13,17-tetramethyloctadecanoyl group.

Regarding the stereochemistry of the branched methyl group,
It may be a racemic body or an optically active body. These can be appropriately selected in consideration of availability of the chain terpene as a raw material, but when an optically active substance is used, (R)
The absolute configuration of is preferred. Such optically active isoprenoids can be obtained by using the method of Noyori et al. (J. Org. Chem., 53, 7) even if a naturally occurring terpene is used as a raw material.
08, 1988, J. Am. Chem. Soc., 109, 1596, 19
It is also possible to prepare by performing asymmetric hydrogenation according to 1987).

X represents a hydrophilic group. However, the hydrophilic group as used herein means a hydrophilic group necessary for the compound represented by the general formula (I) to be in a state of a molecular aggregate such as a liposome or a micelle. Generally, the balance between the new aqueous group and the hydrophobic century in the lipid molecule and the shape of the whole molecule determine the morphology of the molecular assembly in aqueous solution. An index showing the degree of hydrophilicity and hydrophobicity of this amphipathic molecule. As HLB (hydrophilic-lipophilic balance) is known as. Originally, the larger the value of HLB, the stronger the hydrophilicity. Therefore, a substance having a well-balanced hydrophilicity and hydrophobicity forms a stable molecular assembly such as a liposome.

HLB can also be calculated by the following equation. HLB = 11.7 log (Mw / Mo) +7 where Mo = molecular weight of alkyl group Mw = total molecular weight−Mo 2. The balance of the compound of the present invention as an amphipathic molecule can also be defined from this HLB. Preferably, the HLB value is between 2 and 18. HLB is described in detail in the literature (Chemistry, Vol. 7, p. 689, 1953).

As the hydrophilic group X satisfying the above conditions,
Specific examples thereof include a hydrogen atom, choline residue, ethanolamine residue, serine residue, glycerol residue, myo-inositol residue and the like.

The phospholipid used in the present invention may form a salt with a suitable counter ion (including a state of forming a salt in the molecule). However, the salt is preferably physiologically or pharmacologically acceptable. Specifically, salts with inorganic acids such as hydrochlorides, sulfates and nitrates, salts with organic acids such as acetates, trifluoroacetates, lactates, tartrates, ammonium salts, sodium salts and potassium salts. Examples thereof include salts, magnesium salts, calcium salts and the like, and among them, hydrochlorides, acetates, trifluoroacetates and sodium salts are preferable. Conversion to such salts can be accomplished by conventional means.

Specific examples of the phospholipid used in the present invention are shown below, but the present invention is not limited thereto.

[0023]

[Chemical 3]

Next, a method for synthesizing the phospholipid used in the present invention will be described. These phospholipids have typical phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol structures and are found in the literature (eg, AE Stepanov and VISh
vets, Chem. Phys. Lipids 41, 1p, 1980, H. Eib
l, Chem. Phys. Lipids 26, 405, 1980, AE Ste
panov and VI Shvets, Chem. Phys. Lipids 25, 247
Page, 1980, F. Ramirez and JF Marecek, Syntheis
(P. 449, 1985, etc.).

The following are exemplary compounds I-1, I-2, I-3 and I-4 as synthetic examples of the phospholipids used in the present invention.
The synthetic route of is shown. However, the synthetic method is not limited to the one shown here, and various routes are possible. The various protecting groups, solvents and reagents are represented by commonly used abbreviations.

[0026]

[Chemical 4]

[0027]

[Chemical 5]

[0028]

[Chemical 6]

[0029]

[Chemical 7]

Next, a method for producing a liposome using a phospholipid represented by the general formula (I) as a constituent component of a membrane lipid,
Its features and uses will be described. The phospholipid used in the present invention alone can form a stable molecular assembly such as a liposome. This lipid bilayer has a low phase transition point below room temperature because the hydrophobic part linked to the glycerol part is an acyl group having a chain terpene structure, and has fluidity and high mobility similar to those of a biological membrane.

As a method for preparing liposomes, generally known methods are known, for example, a review by Papahajopouls et al.
(Ann. Rev. Biophys. Bioeng., 9: 467, 1980)
Book by Nojima, Sunamoto, Inoue et al. (Liposome, 21-40
P., Nankodo, 1988) is effective. Specific methods for preparing liposomes include a thin film method, an ultrasonic irradiation method, a surfactant removal method, a reverse phase evaporation method, an ethanol injection method, a pre-vesicle method, an extrusion method, and a freeze-thaw method. The method is not limited to these methods, and various combinations are possible. Since the phospholipid used in the present invention has a low phase transition point at room temperature or lower, it has good dispersibility and can be easily formed into a liposome by any method.

The phospholipid used in the present invention can form stable liposomes even when mixed with other lipids. Lipids to be mixed to prepare liposomes include egg yolk, phosphatidylcholine derived from soybean or other animals and plants, phosphatidylethanolamine,
Phosphatidylinositol, phosphatidylserine,
Examples include sphingomyelin, glycosphingolipids such as gangliosides, dipalmitoyl lecithin, distearoyl lecithin, and dimyristoyl lecithin obtained by chemical synthesis. The phospholipid used in the present invention is a material having both high membrane fluidity and mobility, and further chemical stability. Therefore, in order to take advantage of its advantages, chemically stable saturated fatty acid residues are used in the hydrophobic part. It is advantageous to mix with the lipids that they have. Examples of such lipids include phosphatidylcholine having a linear saturated fatty acid residue having 14 to 20 carbon atoms.

There is no particular limitation on the molar ratio at the time of mixing, and it can be mixed with other lipid in the range of 1% to 99% to form a liposome membrane lipid constituent. Preferably,
The proportion of the phospholipid used in the present invention in the total membrane lipid is preferably in the range of 10% to 60%. By changing the mixing ratio of the phospholipid used in the present invention, it is possible to control the membrane fluidity of the lipid bilayer membrane and the membrane barrier ability closely related thereto.

It is also possible to mix dicetylphosphoric acid, stearylamine or the like in order to impart an electric charge to the film.
A molar ratio of 10% or less at the time of mixing is sufficient, and it is effective in preventing aggregation and precipitation of liposomes.

Although steroids such as cholesterol can be mixed with the phospholipid of the present invention, the proportion of steroid in the total membrane lipid is preferably 50% or less, more preferably 30% or less.

Various hydrophilic substances can be included in the liposome prepared as described above. Specific hydrophilic substances include pigments such as carboxyfluorescein, anticancer agents typified by adriamycin and cisplatin, antiviral agents such as interferon, aminoglycosides, β-lactams (eg penicillin). Antibiotics such as insulin, peptide hormones such as insulin, enzyme agents such as lysozyme and asparaginase, immunostimulants such as muramyl dipeptide, immunoglobulins, proteins such as various toxins, etc., but other compounds are used. It is also possible.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0038]

【Example】

Example 1 Synthesis 1 of Exemplified Compound I-1) Synthesis of Intermediate 1 Natural natural diterpene (7R, 11R) -phytol (200 g) was dissolved in ethanol (1000 ml), and platinum oxide (1 g) was added. After that, the reaction mixture was stirred under a hydrogen atmosphere for 6 hours at room temperature. After the reaction was completed, insoluble materials were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 201 g of Intermediate 1 ((3RS, 7R, 11R) -phytanol) as an oil. IR νmax (film) 3340 (br s), 2960 (s), 2930 (s), 2
870 (s), 1465 (s), 1380 (s), 1370 (m), 1060 (s), 73
5 (w) cm-1

2) Synthesis of Intermediate 2 Intermediate 1 (40 g) was treated with carbon tetrachloride: acetonitrile: water =
It was dissolved in a 2: 2: 3 mixed solvent (700 ml), ruthenium trichloride n-hydrate (500 mg) and sodium metaperiodate (70 g) were added thereto, and the reaction mixture was vigorously stirred at room temperature for 4 hours. It was stirred. After completion of the reaction, insoluble substances were removed by filtration through Celite, the filtrate was diluted with methylene chloride, the organic layer was separated, and the aqueous layer was extracted with methylene chloride. The organic layers were combined, washed once with water, and then dried over anhydrous sodium sulfate. Sodium sulfate was filtered off, and the filtrate was concentrated under reduced pressure to give Intermediate 2 ((3RS, 7R, 11R) -phytanic acid) as an oily substance (29 g).
Obtained. IR νmax (film) 3600-2400 (br m), 2960 (s), 2930
(s), 2870 (s), 1715 (s), 1465 (m), 1380 (m), 1370
(w), 1300 (m), 940 (w) cm-1

3) A solution of the synthetic intermediate 2 of I-1 (940 mg) in chloroform (20 ml) was ice-cooled, and N, N-dicyclohexylcarbodiimide (620 mg) and 4-N, N-dimethylamino were added thereto. Pyridine (340
mg) was added and the mixture was stirred for 1 hour. Subsequently, glycerophosphocholine-cadmium chloride complex (640 mg) was added, and the reaction mixture was stirred at room temperature for 4 days. The reaction solution was treated with ion exchange resin Amberlite IR-120B and then purified by silica gel chromatography. Chloroform:
Elution with methanol: water (60: 35: 5) gave the desired I-1 (diphytanoylphosphatidylcholine) as a colorless wax, 270 mg. TLC [using Merck Art 5715 plate, developing solvent: chloroform: methanol: water = 60: 35: 5) Rf
0.54 IR νmax (KBr) 2960 (s), 2930 (s), 2870 (s), 1740
(s), 1380 (s), 1365 (sh), 1250 (s), 1165 (m), 1095
(s), 1065 (s), 970 (s), 820 (m) cm-1 FAB-MS: (M + H) + 846

Example 2 Synthesis of Exemplified Compound I-2 1) Synthesis of Intermediate 3 Thionyl chloride (18 g) was added to a solution of Intermediate 2 (30 g) in toluene (150 ml), and the reaction mixture was stirred for 40 hours. did. After the generation of gas stopped and the reaction was completed, the solvent and excess thionyl chloride were distilled off at atmospheric pressure. The residue was dried under reduced pressure to obtain 32 g of the intended intermediate 3 ((3RS, 7R, 11R) -phytanoyl chloride) as an oil. IR νmax (film) 2960 (s), 2930 (s), 2870 (s), 180
0 (s), 1465 (s), 1380 (s), 1370 (m), 990 (m), 825
(s) cm-1

2) Synthesis of Intermediate 4 3-Benzyl-sn-glycerol (5.4 g, literature [Synthesis
503, 1985] and a solution of diisopropylethylamine (10 g) in methylene chloride (100 ml), a solution of intermediate 3 (22.1 g) in methylene chloride (50 ml) is added, and the reaction mixture is added. The mixture was stirred at room temperature for 20 hours in the presence of a catalytic amount of 4-N, N-dimethylaminopyridine. The reaction mixture was washed with water and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (eluent hexane / ethyl acetate = 20/1),
19 g of Intermediate 4 was obtained as an oily substance. IR νmax (film) 3030 (w), 2960 (s), 2930 (s), 2870
(s), 1745 (s), 1500 (w), 1460 (s), 1380 (s), 1370
(m), 1245 (m), 1165 (s), 1120 (s), 1110 (sh), 730
(m), 695 (s) cm-1

3) Synthesis of Intermediate 5 To a solution of Intermediate 4 (10 g) in ethyl acetate (200 ml) was added 5% palladium-carbon (1 g), and the reaction mixture was stirred under a hydrogen atmosphere at room temperature for 6 hours. After the reaction was completed, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 8.6 g of the target Intermediate 5 as an oily substance. IR νmax (film) 3460 (br m), 2950 (s), 2920 (s), 2
870 (s), 1745 (s), 1465 (s), 1380 (s), 1370 (sh), 1
240 (m), 1165 (s), 1130 (m), 1100 (w), 1045 (m) cm-
1 Elemental analysis calculated: C43H84O5: C, 75.88; H, 12.35%. Found: C, 75.82; H, 12.30%.

4) Synthesis of Intermediate 6 Intermediate 5 (5 g) and diisopropylethylamine (1.15
To a solution of g) in methylene chloride (50 ml) was added diphenylphosphorochloridate (2.4 g) and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, dilute with methylene chloride,
The extract was washed with water, saturated sodium hydrogen carbonate solution and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain Intermediate 6 as an oily substance in an amount of 5.2 g. IR νmax (film) 3060 (w), 2950 (s), 2930 (s), 2860
(s), 1745 (s), 1595 (s), 1490 (s), 1460 (s), 1380
(m), 1370 (sh), 1290 (m), 1190 (s), 1160 (m), 1060
(m), 960 (s), 755 (s), 735 (m), 685 (m) cm-1

5) Platinum oxide (100 mg) was added to a solution of the synthetic intermediate 6 of I-2 (5.2 g) in ethyl acetate (60 ml), and the reaction mixture was stirred under a hydrogen atmosphere at room temperature for 8 hours. After completion of the reaction, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain the desired I-2 (diphytanoylphosphatidic acid) as a colorless transparent oil (4.3 g). IR νmax (film) 3600-2000 (br m), 2950 (s), 2920
(s), 2860 (s), 1745 (s), 1460 (s), 1380 (s), 1370
(sh), 1240 (s), 1165 (s), 1060 (s), 1020 (s) cm-1 Elemental analysis calculated: C43H85O8P: C, 67.89; H, 11.18%. Found: C, 67.43; H , 11.33%.

Example 3 Synthesis of Exemplified Compound I-3 1) Synthesis of Intermediate 7 In a solution of phenylphosphorodichloridate (1.37 g) in tetrahydrofuran (15 ml), Intermediate 5 (4.0 g) and N-methylimidazole ( 530 mg) of tetrahydrofuran (20
ml) solution was added at room temperature and the reaction mixture was stirred for 30 minutes. Thereafter, a solution of Nt-butoxycarbonylethanolamine (1.0 g) and N-methylimidazole (530 mg) in tetrahydrofuran (20 ml) was added, and the reaction mixture was stirred for 2 hours. The reaction solution was poured into 100 ml of water and extracted with ethyl acetate. The organic layers were combined, washed with water, aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a colorless oil. This product was purified by silica gel chromatography (eluent hexane / ethyl acetate = 20/1 to 8/1) to obtain 2.89 g (yield 50.0%) of Intermediate 7 as a colorless oil. IR νmax (film) 3370 (m), 2950 (s), 2930 (s), 2860
(s), 1745 (s), 1720 (s), 1595 (m), 1380 (sh), 1370
(s), 1245 (s), 1165 (s), 1040 (s), 755 (s), 680
(w) cm-1 Elemental analysis calculated: C56H102NO10P: C, 68.64; H, 10.42
%. Found: C, 68.37; H, 10.78%.

2) To a solution of the synthetic intermediate 7 of I-3 (2.3 g) in methylene chloride (15 ml) was added trifluoroacetic acid (15 ml), and the reaction mixture was stirred at room temperature for 40 minutes.
Stir for minutes. After confirming the completion of the reaction, the solvent was removed by concentration under reduced pressure to quantitatively obtain the de-t-Boc form. This was dissolved in ethyl acetate (30 ml), 50 mg of platinum oxide was added, and the reaction mixture was stirred under a hydrogen atmosphere for 20 hours. The insoluble material was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain 1.9 g of the target I-3 (diphytanoylphosphatidylethanolamine) as a viscous oil. IR νmax (film) 3000-2200 (br m), 2960 (s), 2930
(s), 2860 (s), 1745 (s), 1620 (s), 1460 (s), 1380
(m), 1370 (sh), 1210 (s), 1155 (s) cm-1 FAB-MS: (M + Na) + 826

Example 4 Synthesis of Exemplified Compound I-4 1) Synthesis of Intermediate 8 I-2 (diphytanoylphosphatidic acid, 2 g) and Z-serine benzyl ester (900 mg, usually from Z-serine and benzyl bromide) (Prepared by the method), dry pyridine was added, and the mixture was dried azeotropically. This was dissolved in dry pyridine (10 ml), 2,4,6-trimethylbenzenesulfonyl chloride (900 mg) was added, and the reaction mixture was stirred at room temperature for 15 hours. The reaction was stopped by adding an appropriate amount of ice, and the mixture was extracted with chloroform. The organic layer was washed with water, 1N citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. This product was purified by silica gel chromatography (eluent: methylene chloride / methanol = 20/1) to obtain Intermediate 8 as an oil. IR νmax (film) 3600-2200 (br m), 2960 (s), 2930
(s), 2860 (s), 1745 (s), 1720 (s), 1600 (m), 1460
(s), 1380 (s), 1370 (sh), 1255 (s), 1160 (s), 1035
(s), 740 (m), 695 (m) cm-1

2) 10% Pd-C (100 mg) as a catalyst was added to a solution of the synthetic intermediate 9 of I-4 (5.2 g) in ethyl acetate (60 ml), and the reaction mixture was heated under hydrogen atmosphere at room temperature for 8 hours. It was stirred. After completion of the reaction, insoluble substances were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure to obtain the desired I-4 (diphytanoylphosphatidylserine) as a viscous oil. IR νmax (film) 3550-2000 (br m), 2960 (s), 2930
(s), 2860 (s), 1745 (s), 1720 (s), 1610 (s), 1460
(s), 1380 (s), 1370 (sh), 1220 (s), 1130 (s) cm-1 FAB-MS: (M + Na) + 870

Example 5 Synthesis of Exemplified Compound I-5 Using intermediate 5 and 1,2-O-isopropylidene glycerol as starting materials, phosphoric esterification and subsequent deprotection were carried out by the method described in Example 3. , The target I-5 is 98
0 mg was obtained. IR νmax (film) 3400-2150 (br m), 2960 (s), 2930
(s), 2860 (s), 1745 (s), 1460 (s), 1380 (s), 1370
(sh), 1250 (s), 1210 (s), 1150 (m), 1060 (s) cm-1 FAB-MS: (M + Na) + 857

Example 6 Synthesis of Exemplified Compound I-6 Using intermediate 5 and tetra-O-benzyl-myo-inositol as starting materials, the literature (VIShvets et al., Tetrahedr
on Vol. 29, p. 331, 1973), phosphoric esterification and subsequent deprotection were carried out to obtain 280 mg of the target I-6 as a colorless powder. IR νmax (Nujol) 3350-2000 (br m), 2960 (s), 2940
(s), 2860 (s), 1745 (s), 1460 (s), 1380 (s), 1370
(sh), 1245 (s), 1220 (s), 1120 (s) cm-1 Elemental analysis calculated: C49H95O13P.H2O: C, 62.55; H, 10.3
2%. Found: C, 62.03; H, 10.49%.

Example 7 Results of Evaluation of Thermophysical Properties of Exemplified Compounds I-1 to I-6 The phospholipids used in the present invention were concentrated at a concentration of 0.1% in pure water using a Pivarov type adiabatic differential scanning microcalorimeter DASM-4. The phase transition temperature was measured. As a result, the exemplary compound I-
It was found that each of 1 to 1-6 had a phase transition temperature of 2 ° C. or lower.

Example 8 Evaluation Results of Thermophysical Properties in a Mixing System of Dipalmitoylphosphatidylcholine (DPPC) and Exemplified Compound I-1 Exemplified Compound I-1 and DPPC were mixed at various molar ratios,
The phase transition temperature was measured by the same method as in Example 7. The result is shown in FIG. From this, the exemplary compound I of the present invention
It can be seen that -1 (diphytanoylphosphatidylcholine) has a phase transition temperature elimination ability similar to that of cholesterol for DPPC having a linear saturated fatty acid residue and enhances the fluidity of the lipid bilayer membrane.

Example 9 Preparation of liposome by thin film method using Exemplified Compound I-1 Exemplified Compound I-1 (30 mg) was dissolved in chloroform (20 ml) and concentrated under reduced pressure to form a thin film. After sufficiently drying in a nitrogen atmosphere, 3 ml of a buffer solution (6 mM Tris-HCl, pH 7.4) was added, and vortex mixing was performed for 10 minutes to roughly disperse the mixture. Then, sonication was performed for 15 minutes with a probe-type ultrasonic generator to obtain a liposome dispersion liquid.

As a result of measuring the average particle size, the obtained liposome dispersion liquid had a diameter of 240 nm, and was confirmed to be an oligolamellar vesicle by morphological observation with an electron microscope. Further, the particles were allowed to stand at room temperature for 20 days, and then the particle size was measured, but no particular change was observed, and neither aggregation nor precipitate was observed.

Example 10 Method for preparing carboxyfluorescein-encapsulating liposomes by the thin film method using Exemplified Compound I-1 and leakage test of the inclusion compound Exemplified Compound I-1 (30 mg) was dissolved in chloroform (20 ml) and the pressure was reduced. Concentrated to form a thin film. After thoroughly drying in a nitrogen atmosphere, a carboxyfluorescein buffer solution (20
0 ml / 6 mM Tris-HCl, pH 7.4, containing 150 mM NaCl) 3 ml
Was added, and vortex mixing was performed for 15 minutes for coarse dispersion. Then, sonication was performed for 10 minutes with a probe-type ultrasonic generator to obtain a liposome dispersion liquid. The non-encapsulated carboxyfluorescein was removed by gel filtration chromatography to obtain a liposome fraction encapsulating carboxyfluorescein.

After measuring the particle size of liposomes in the obtained fraction, a part having an average particle size of 200 to 300 nm was selected, and 6 mM Tris-HCl buffer solution (pH 7.4) containing 150 mM NaCl was selected.
The lipid concentration was adjusted to 2 × 10 -4 M by diluting with and the leakage of carboxyfluorescein encapsulated at various temperatures was monitored by fluorescence measurement. The time-dependent change in leakage is shown in FIG.

Example 11 Method for preparing carboxyfluorescein-encapsulating liposome in mixed system of dipalmitoylphosphatidylcholine (DPPC) and Exemplified compound I-2 and leakage test of inclusion compound DPPC (20 mg) and Exemplified compound I-2 (10 mg) Was dissolved in chloroform (20 ml) and concentrated under reduced pressure to form a thin film. The same procedure as in Example 10 was performed below to obtain a fraction of liposomes containing carboxyfluorescein, and the leakage of carboxyfluorescein encapsulated at various temperatures was monitored by fluorescence measurement. The change in leakage over time is shown in FIG.

Comparative Example 1 Preparation of carboxyfluorescein-encapsulating liposome in dipalmitoylphosphatidylcholine (DPPC) alone system and its content leakage test DPPC (30 mg) was carried out in the same manner as in Example 10,
A carboxyfluorescein-encapsulated liposome was obtained by the operation, and the leakage of carboxyfluorescein encapsulated at various temperatures was traced by fluorescence measurement. Figure 4 shows the change over time of leakage
Shown in.

Comparative Example 2 Preparation of carboxyfluorescein-encapsulating liposomes in a mixed system of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) and the leakage test of inclusion bodies DPPC (20 mg) and DPPA (10 mg) were used. Then, carboxyfluorescein-encapsulated liposomes were obtained by the same method and operation as in Example 11, and the leakage of carboxyfluorescein encapsulated at various temperatures was traced by fluorescence measurement.
The time-dependent change of leakage is shown in FIG.

Comparative Example 3 Preparation of carboxyfluorescein-encapsulating liposome in a mixed system of dipalmitoylphosphatidylcholine (DPPC) and cholesterol and leakage test of its encapsulation Same as Example 11 using DPPC (20 mg) and cholesterol (10 mg). Carboxyfluorescein-encapsulated liposomes were obtained by the method and operation described in 1. above, and the leakage of carboxyfluorescein encapsulated at various temperatures was monitored by fluorescence measurement. The time course of leakage is shown in FIG.

Example 12 Results of Acute Toxicity Test of Phospholipids Used in the Present Invention in Mice (1) Exemplified Compounds I-1 (Diphytanoylphosphatidylcholine) and I-2 (Diphytanoylphosphatidic acid) .
Each of these was dispersed in PBS at a concentration of 10 mg / ml to prepare a test solution.

(2) Animals used and breeding environment Male Slc: ICR mice aged 4 weeks were used for the test after being kept in the environment for 6 days. When used, the age is 5 weeks and the weight is
It was around 25 g. The animals are pulp bedding (Alpha Dry: manufactured by SSP) in an animal room in a breeding room (temperature: 23 ° C, humidity: 50%, ventilation rate: 21 times / hour, lighting time: 12 hours) with automatic environmental control. Each of them was housed in a flat-bottomed polycarbonate cage having 4 cages, and water and solid feed (MF: manufactured by Oriental Yeast) were allowed to freely ingest.

(3) Test method The test solution was intravenously administered through the tail vein, and a limit test of 20 ml / kg was carried out using 4 animals per group. After the administration, observation of the general form was performed immediately after the administration for 8 hours and from the next day for 14 days every day, and the measurement of body weight and feed intake was 1, 2, 3, 7, 14
Went a day later. In addition, dead animals were dissected promptly after discovery, and surviving animals were dissected 14 days after administration, and macroscopic observation of various organs was performed.

(4) Test Results No deaths were observed when both the exemplified compounds I-1 and I-2 were administered at a dose of 20 ml / kg. There were no notable changes in the symptoms of intoxication, and the dietary intake and weight gain were considered to be normal. There were no significant changes in anatomical findings.

Example 13 Action of Liposomes Formed by Phospholipids Used in the Present Invention on Lymphocytes (1) Test Compound Exemplified Compound I-1 (30 mg) was dissolved in 20 ml of chloroform and concentrated under reduced pressure. A thin film was formed. After sufficiently drying under reduced pressure,
3 ml of physiological saline was added, and the mixture was roughly dispersed by a vortex mixer. Subsequently, an extruder (using a 0.2 μm polycarbonate filter) was used to obtain a liposome dispersion liquid having a uniform particle diameter of about 0.2 μm.

For comparison, liposomes were similarly prepared for dimyristoylphosphatidylcholine (DMPC). (2) Test method (spleen cell proliferation test by mitogen) Mouse splenocytes were collected and the number of cells with a size of 5 to 9 μm was 4
RPMI containing 10% fetal bovine serum so that the number becomes 106
A cell suspension is prepared in 1640 medium.

100 μl of the cell suspension was dispensed into a flat-bottom 96-well microplate, and the substance to be tested was 0.02 to 20 μg / 50 μl R
PMI1640 medium and Concanavalin A 0.4 μg / 50 μl RPMI1640 medium as mitogen are added to each well. Incubate for 2 days in a CO2 incubator, and add 0.4 µCi of 3 [H] thymidine 6 hours before the end of the culture.
Add / 20 μl to each well. After completion of the culture, the cells are collected on a glass fiber filter using a cell harvester, dried, and transferred to a scintillation vial. Toluene scintillator is added to a vial, and thymidine ingested into cells is measured by a liquid scintillation counter.

In the same manner, 50 μl of the cell culture solution which had been cultured for 2 days was taken, and 0.15% trypan blue physiological saline solution was added thereto. Cell viability is determined by counting the number of rapidly stained cells (dead cells) and the number of unstained cells (live cells) under a microscope. (3) Test Results Splenocytes divide and grow by the stimulation of concanavalin A, but it is well known that the growth is inhibited when liposomes composed of phospholipid coexist. (Eg Bioche
m. Biophys. Res. Commun., 79,852 (1977)) The result of cell viability is shown in FIG. In either case of the two types of liposomes, the survival rate decreases as the concentration increases, but it can be seen that Exemplified Compound I-1 has a higher survival rate than DMPC and is less cytotoxic. In addition, phospholipids such as DMPC inhibit the cell proliferation response to mitogen (FIG. 8), but Exemplified Compound I-1 is much less.

From these results, it is clear that Exemplified Compound I-1 forms liposomes with less cytotoxicity and less inhibition of cell function than similar phospholipids having a linear alkyl group.

[0071]

By using the liposome of the present invention, the following remarkable effects can be obtained. (1) The phospholipid used in the present invention is easy to synthesize because the method of connecting the glycerol moiety and the hydrocarbon chain is an ester bond rather than the ether type found in archaeal biomembrane lipids, and is particularly suitable for large-scale synthesis. Very advantageous. (2) Since the chain isoprenoid used as the raw material for synthesizing the phospholipid used in the present invention has a reliable structure and stereochemistry as compared with isoacid and antiisoacid, it can be easily obtained.
From this point as well, it is advantageous for synthesis. (3) The phospholipid used in the present invention has a low phase transition point below room temperature because the hydrophobic portion linked to the glycerol portion is a saturated acyl group having a chain terpene structure. It has properties that cannot be achieved by the following phospholipids or phospholipids having unsaturated fatty acids, that is, excellent dispersibility, good membrane fluidity, and particularly excellent chemical stability. Therefore, the bilayer membrane having phospholipid as a membrane lipid component used in the present invention, specifically liposome, has membrane fluidity extremely close to that of a biological membrane, and is useful as a biological membrane model in the field of biomembrane mimetics research. It is a thing. (4) Furthermore, the bilayer membrane of the present invention containing the phospholipid as a membrane lipid constituent component, specifically, the liposome is described in Examples 10 and 1.
1. As is clear from Comparative Examples 1 to 3, it exhibits extremely high ability to suppress leakage of inclusions, and its effect is particularly remarkable in the high temperature range of 40 ° C. or higher as compared with the conventionally used liposomes. Such high membrane barrier ability, that is, water-soluble substance-retaining ability is particularly useful when liposomes are used as drug carriers. (5) Further, from Example 8, when mixed with DPPC having a linear saturated fatty acid residue, the phospholipid used in the present invention has a phase transition temperature elimination ability similar to that of cholesterol, and the lipid bilayer membrane flows. You can see that it enhances sex. By utilizing this property, it is possible to freely control the membrane fluidity of the liposome and the membrane barrier ability closely related to it by changing the mixing ratio of the phospholipid used in the present invention. (6) The phospholipid used in the present invention and the liposome formed therefrom have almost no problem of toxicity to living tissues and cells as is clear from Examples 12 and 13, and also have an inhibitory effect on cell function. Is also small.
Therefore, when the liposome of the present invention is used as a drug carrier, it has less effect on cells and blood cells around the administration site, and is advantageous also from this point.

[Brief description of drawings]

1 shows DPPC and exemplified compound I- described in Example 8. FIG.
It is a graph which shows a DSC curve when 1 is mixed in various ratios.

FIG. 2 is a graph showing a time-dependent change in leakage of encapsulated carboxyfluorescein at various temperatures from a liposome containing Exemplified Compound I-1 as a membrane lipid component described in Example 10. Leakage rate (%) on the vertical axis, time (minutes) on the horizontal axis
Represents

FIG. 3 DPPC and Exemplified Compound I described in Example 11.
2 is a graph showing the time-dependent change in the leakage of carboxyfluorescein encapsulated in liposomes composed of a mixed system of -2 at various temperatures. The vertical axis represents the leakage rate (%), and the horizontal axis represents the time (minutes).

FIG. 4 is a graph showing a time-dependent change in leakage of carboxyfluorescein encapsulated in liposomes described in Comparative Example 1 and containing DPPC alone as a membrane lipid constituent at various temperatures. The vertical axis represents the leakage rate (%), and the horizontal axis represents the time (minutes).

FIG. 5 is a graph showing a time-dependent change in leakage of encapsulated carboxyfluorescein from liposomes containing DPPC and DPPA as membrane lipid constituents described in Comparative Example 2 at various temperatures. The vertical axis represents the leakage rate (%), and the horizontal axis represents the time (minutes).

FIG. 6 is a graph showing a time-dependent change in leakage of encapsulated carboxyfluorescein at various temperatures from a liposome described in Comparative Example 3 and containing DPPC and cholesterol as membrane lipid constituents. The vertical axis represents the leakage rate (%), and the horizontal axis represents the time (minutes).

7 is a graph showing the relationship between liposome concentration and cell viability under concanavalin A-stimulated conditions described in Example 13. FIG. The vertical axis represents cell viability (%), and the horizontal axis represents liposome concentration (μg / ml).

FIG. 8 is a graph showing the effect of concanavalin A stimulation on cell proliferation in the presence of liposomes described in Example 13. The vertical axis represents the amount of thymidine uptake (× 104 DP
M indicates the degree of cell proliferation), and the horizontal axis indicates the liposome concentration (μg / ml).

Claims (2)

[Claims] 1. A liposome vesicle comprising a phospholipid represented by the following general formula (I) as a membrane lipid component. General formula (I) (In the formula, R represents an acyl group having a chain terpene structure having 10 to 25 carbon atoms and a plurality of methyl groups. X represents a hydrophilic group. Here, the hydrophilic group means a general formula ( The compound represented by I) means a hydrophilic group necessary for forming a molecular aggregate state such as liposomes and micelles, and the ionic group present in the molecule is a suitable counterion and salt. (Including the state of forming) 2. X in the phospholipid represented by the general formula (I) is selected from the group consisting of choline residue, hydrogen atom, ethanolamine residue, glycerol residue, serine residue and myo-inositol residue. The liposome vesicle according to claim 1, which is at least one selected.