JP2700276B2 - Liposome - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to liposomes, and in particular, to liposomes that avoid uptake by reticuloendothelial system (RES) tissue and can be present in blood at high concentrations for extended periods of time.

BACKGROUND ART Liposomes are closed vesicles of a lipid membrane mainly composed of phospholipids, which can be used for physicochemical research as a model of a biological membrane or can hold various substances in an inner water layer or a membrane. Research has been actively conducted as one of drug delivery systems holding various drugs. As the transport system, for example, peppers, b. E. [Academic Science (Acad.Sci.) 308,281]
(1978)], Gregorian jizy. [Liposome Technology CRL Press Inc. (Li
posome Technology CRCP ress Inc.)], Weinstein J. N's report [Science 20
4,188 (1979)]. It is particularly effective in reducing the toxicity of highly toxic drugs, stabilizing drugs that are unstable in the body, sustained release of drugs in vivo, and utilizing the properties of being fused or taken up by cells. Therefore, it is also known that it can be used as a means for selectively transferring a drug to specific cells. Specific applications include, for example, enzymes (superoxide dismutase), drugs (particularly antitumor agents, adriamycin), chelating agents, hormonal agents (steroid compounds), radionuclides, interferons, interleukins, antigens, It is known to form antibodies into liposomes. In addition, a drug with a short half-life in blood, such as insulin, is encapsulated in the liposome to enhance the durability of the drug, or a cancer surface-specific antibody is bound to the liposome containing the anticancer drug to select cancer cells. Attacking therapy (missile therapy) has been developed.

However, in the case of in vivo use, the liposome administered to the living body is captured by the reticuloendothelial tissue in a short time. This capture is a result of the recognition of the liposome, which is a polymer, as a foreign substance. Such a property is a major obstacle when aiming at directing liposomes to tissues other than RES or for enhancing the persistence of drug efficacy in blood.

In order to solve such a defect, a liposome containing sialic acid in a lipid bilayer has been proposed (US Pat. No. 4,501,728). However, these liposomes also cannot sufficiently avoid the capture by RES, and the effect of maintaining the drug effect in blood is not satisfactory.

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a liposome which is hardly captured by RES and can be present in a blood at a high concentration for a long time.

Other objects and features of the present invention will become apparent from the following description.

That is, the present invention provides, in the lipid membrane of the liposome, uronic acid, uronic acid oligosaccharide, oligosaccharide composed of glucosamine and glucuronic acid, oligosaccharide composed of galactosamine and glucuronic acid, oligosaccharide composed of glucuronic acid and xylose, glucuronic acid and glucose. Oligosaccharide consisting of glucuronic acid and galactose, oligosaccharide consisting of glucuronic acid and mannose, oligosaccharide consisting of galacturonic acid and rhamnose, ketoaldonic acid, 2-acetamido-2-deoxyuronic acid, 2-acetamido-2 -Deoxyuronic acid oligosaccharide, 6-O-carboxyethyl-
β-D-glucose, 6-O-carboxymethyl-β-
D-glucose, 6'-O-carboxymethyl-β-D
-Maltose and 6'-O-carboxyethyl-β-D
-A liposome characterized by containing a glycolipid comprising at least one sugar selected from the group consisting of maltose and a lipid formed by an O-glycosidic bond or an S-glucosidic bond.

According to the study of the present inventors, a liposome in which a specific glycolipid other than sialic acid is incorporated into the lipid membrane is difficult to be captured by RES, and can be present in blood at a high concentration for a long time, and By using a liposome having such a remarkable effect, it has been found that an unprecedentedly high blood concentration is maintained for a long time, and it is extremely useful for pharmaceutical uses such as anticancer drugs, enzyme preparations, proteins, peptide preparations, and the like. The present invention has been completed.

In the present invention, the glycolipid incorporated into the lipid bilayer membrane of the liposome is a sugar and a lipid shown below, which are formed by an O-glycoside bond or an S-glycoside bond.

Examples of the saccharide include uronic acid, uronic acid oligosaccharide, oligosaccharide composed of glucosamine and glucuronic acid, oligosaccharide composed of galactosamine and glucuronic acid, oligosaccharide composed of glucuronic acid and xylose, oligosaccharide composed of glucuronic acid and glucose, and glucuronic acid. Oligosaccharides consisting of and galactose, oligosaccharides consisting of glucuronic acid and mannose, oligosaccharides consisting of galacturonic acid and rhamnose, ketoaldonic acid, 2-acetamido-2-deoxyuronic acid, 2-acetamido-2-deoxyuronic acid oligosaccharides, 6-O-carboxyethyl-β-D-glucose,
6-O-carboxymethyl-β-D-glucose, 6 ′
-O-carboxymethyl-β-D-maltose and 6 ′
At least one selected from the group consisting of -O-carboxyethyl-β-D-maltose can be used. These sugars are all monosaccharides or disaccharides having at least one carboxyl group on the sugar chain.

Examples of uronic acids include L-iduronic acid, D-galacturonic acid, D-glucuronic acid, L-glucuronic acid,
-Mannuronic acid and the like.

Examples of uronic acid oligosaccharides include 2-β-glucuronosyl glucuronic acid, α-1, -4′-galacturonosyl galacturonic acid, 4-β-mannuronosyl mannuronic acid, and 2-β- Glucuronosyl glucuronic acid and the like can be mentioned.

Examples of the oligosaccharide comprising glucosamine and glucuronic acid include hyalobiouronic acid, heparosin and the like.

Examples of the oligosaccharide composed of galactosamine and glucuronic acid include chondrosin.

Oligosaccharides composed of glucuronic acid and xylose include, for example, 4-β-glucuronoxylose, 3-α-
Glucuronoxylose, 2-α-glucuronoxylose, 2-α-4-O-methylglucuronoxylose, 3
-Α-4-O-methylglucuronoxylose and the like.

As an oligosaccharide composed of glucuronic acid and glucose, for example, cellobiouronic acid and the like can be mentioned.

Examples of the oligosaccharide composed of glucuronic acid and galactose include 6-β-glucuronogalactose.

Examples of the oligosaccharide composed of glucuronic acid and mannose include, for example, 2-β-glucuronomannose.

Examples of the oligosaccharide composed of galacturonic acid and rhamnose include 2-α-galacturono-L-rhamnose.

Examples of ketoaldonic acid include D-arabinose-hexuronic acid.

6-O-carboxyethyl-β-D-glucose is
For example, 1,2,3,4 obtained by benzylating glucose
-Tetra-O-benzyl-β-D-glucose is reacted with succinic anhydride and then debenzylated.

6-O-carboxymethyl-β-D-glucose is
For example, it is produced by reacting 1,2,3,4-tetrabenzyl-β-D-glucose with methyl bromoacetate in the presence of sodium hydride, followed by debenzylation by hydrolysis and catalytic reduction of methyl ester. it can.

6'-O-Carboxymethyl β-D-maltose is, for example, 1,2,2 obtained by benzylation of maltose.
It can be produced by reacting 3,6,2 ', 3', 4'-heptabenzyl maltose with ethyl bromoacetate in the presence of sodium hydride, followed by hydrolysis with ethyl ester and debenzylation by catalytic reduction.

Among the above saccharides, uronic acid, uronic acid oligosaccharide, oligosaccharide composed of glucosamine and glucuronic acid, oligosaccharide composed of galactosamine and glucuronic acid, oligosaccharide composed of glucuronic acid and galactose, oligosaccharide composed of glucuronic acid and mannose, and the like are included. Preferably, further, uronic acid,
Particularly preferred are uronic acid oligosaccharides, oligosaccharides composed of glucosamine and glucuronic acid, oligosaccharides composed of galactosamine and glucuronic acid, and the like.

Known lipids can be used as the lipids that form O- or S-glycosidic bonds with the sugars to form glycolipids. Among them, diacylglycerides, dialkylglycerides, sphingosine, ceramide, hydrocarbons and cholesterol can be used. At least one of them is preferred.

Examples of diacyl glycerides include, for example, 1,2-dipalmitoyl glyceride, 1,2-distearoyl glyceride, 1,2-dioleoyl glyceride, and the like, in which the acyl moiety is a saturated or unsaturated fatty acid having about 10 to 40 carbon atoms. Examples thereof include 1,2-diacylglycerides which are acyl groups.

As dialkyl glycerides, for example, 1,2-
1,2-O-dialkyl glycerides in which the alkyl portion is a linear or branched alkyl group having about 10 to 40 carbon atoms, such as O-dihexadecyl glyceride and 1,2-O-dioctadecyl glyceride. be able to.

Examples of the hydrocarbon include a saturated or unsaturated linear or branched aliphatic hydrocarbon having about 10 to 60 carbon atoms, preferably about 14 to 40 carbon atoms. The above lipids can be used alone or in combination of two or more.

The glycolipid used in the present invention can be produced by subjecting the above sugar and lipid to a known glycosidation method such as the Koenig-Knoll method.

In the present invention, known lipids other than glycolipids, such as phospholipids and cholesterol, are used as the liposome membrane components together with the glycolipids described above. Any known phospholipids can be used, and specific examples thereof include, for example, soybean reticin, egg yolk lecithin, dipartoylphosphatidylcholine, distearoylphosphatidylcholine, dioleylphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, Examples include phospholipids such as phosphatidylinositol and hydrogenated lecithin and other natural phospholipids that have been hydrogenated. Cholesterol and charged phospholipids such as phosphatidic acid, phosphatidylserine, phosphatidylglycerol, cardiopyrine and the like may be used together with other phospholipids to further prevent liposome fusion and leakage of the encapsulated material. The above phospholipids can be used alone or in combination of two or more.

In the present invention, if necessary, as a liposome membrane component, in addition to the above lipids, a linear or branched saturated or unsaturated fatty acid having about 10 to 40 carbon atoms, diglyceride, triglyceride, cholesterol ester of the fatty acid Etc. may be used as appropriate.

The liposome of the present invention can be in the form of a known liposome. Specific examples thereof include, for example, multilamellar liposomes (MLV; multilamellar vesicles), small unilamellar liposomes (SUVs), and large unilamellar liposomes (LUV; large unilamellar vesicl).
e) and the like.

The liposome of the present invention can be produced according to a known method.
An example is given below.

First, the phospholipid, the glycolipid of the present invention, and, if necessary, cholesterol are dissolved in an appropriate solvent. Next, the obtained solution is put into, for example, a rotary evaporator to distill off the solvent, and a lipid film is formed on the inner wall surface of the evaporator. Add an aqueous solution or buffer solution of the enclosed material into this,
By vigorously shaking, a dispersion of the liposome of the present invention can be obtained. After shaking, freeze-thaw sonication may be performed if necessary. The liposome particle size may be sized using a polycarbonate film.

The amount of the phospholipid used is not particularly limited, and may be appropriately selected depending on the type of the enclosed material to be used and the like. The amount of glycolipid used is not particularly limited, but considering the ease of formation of liposomes, the glycolipid is blended so as to contain about 1 to 50 mol%, preferably about 5 to 30 mol% in the lipid membrane of the glycolipid. do it.
The encapsulated material may be appropriately selected from known encapsulated materials for liposomes such as drugs and markers, depending on the intended use of the liposome. The amount of the enclosed material is not particularly limited, and may be appropriately selected. As the suitable solvent, any solvent capable of dissolving the lipid to be used can be used.For example, halogenated hydrocarbons such as chloroform, methyl chloroform, methylene chloride, hydrocarbons such as hexane and heptane, benzene, toluene, xylene And ethers such as diethyl ether, diisopropyl ether, ethylene glycol dimethyl ether and tetrahydrofuran. The temperature conditions when the solvent is distilled off to form a lipid film are not particularly limited.
The temperature can be appropriately selected from a wide range of about 100 ° C, but is preferably about 25 to 60 ° C, more preferably about 30 to 40 ° C in consideration of preventing lipid oxidation. The pH and salt concentration at the time of liposome preparation are not particularly limited as long as denaturation of lipids and liposomes does not occur.
Degree, osmolarity about 0.3.

By purifying the obtained liposome dispersion according to a known method such as gel filtration and centrifugation, it is possible to separate the liposome from a drug, a marker, and the like not encapsulated in the liposome. By adding a buffered aqueous solution (Tris, phosphoric acid, HEPES, MOPS, etc.) prepared to be isotonic to the liposome-encapsulated solution to the separated liposomes, the suspension of the liposome of the present invention containing the above specific glycolipid is added. A suspension is obtained. The fact that the liposome of the present invention can be obtained by the above-described operation means that the liposome obtained by the same operation as described above in Examples described below can exist in blood at a high concentration for a long time, and is hardly captured by RES. Is also apparent from the above.

 Although the particle size of the liposome of the present invention is not particularly limited, it is usually about 0.03 to 0.8 μm, preferably about 0.05 to 0.5 μm.

Examples Hereinafter, the present invention will be described in detail based on examples.

Reference Example 1 (Synthesis of glycolipid) Glucuronolactone (manufactured by Hanoi Chemical Co., Ltd.) was reacted in methanol in the presence of a catalytic amount of sodium methylate to obtain methyl D-glucopyranosyl uronate. This is acetylated with acetic anhydride in pyridine,
Methyl (2,3,4-tri-O-acetyl-β-D-glycopyranosyluronate, which was further brominated in hydrogen bromide saturated acetic acid to give methyl (2,3,4-tri- O-acetyl-α-D-glucopyranosyl promido) uronate was synthesized and reacted with palmityl alcohol at 25 ° C. in the presence of silver carbonate, and the product was recrystallized from ethanol to give methyl (hexadecyl 2,3, 4-Tri-O-acetyl-β-D-glycopyranosyl) uronate was obtained in a yield of 50.
% (From glucuronolactone). This was further deacetylated with a catalytic amount of sodium methylate in methanol and dried. The obtained residue was suspended in a 50% aqueous methanol solution, and the resulting mixture was subjected to ester hydrolysis by adding potassium hydroxide, and then adjusted to pH 3 with dilute hydrochloric acid.
Extraction with methanol (2/1) yielded hexadecyl β-D-glucopyranosyluronic acid (referred to as glycolipid A) as pale yellow crystals in a yield of 30% (from glucuronolactone).

Example 1 In order to use the liposome obtained in this example for avoidance of the reticuloendothelial system and a blood concentration test described below, tripalmitin labeled with ¹⁴ C was used as one of the liposome membrane components, and as a liposome-encapsulated material. ^{Using 3} H-inulin, liposomes were produced as follows.

Dissolve 80 μmol of dipalmitoylphosphatidylcholine (DPPC), 80 μmol of cholesterol and 40 μmol of glycolipid A in 20 ml of chloroform: methanol = 2: 1 (v: v) and add ¹⁴ C-tripalmine 30 μci to the eggplant-shaped flask. After the addition, the solvent was distilled off using a rotary evaporator. 0.79 ml of sterilized distilled water and 200 μci (0.2 ml) of ³ H inulin were added to the film formed in the flask, and the mixture was stirred with a vortex mixer until the film was peeled off. After the obtained emulsion was frozen with liquid nitrogen, it was dissolved in warm water at 40 ° C. After this operation was repeated twice, 0.13 ml of sterilized 3M glycol was added, and the freeze-thawing operation was performed twice. The resulting emulsion was passed twice through a 0.1 μm membrane filter. 3000 solution
Centrifuge at 5 rpm for 5 minutes, remove the upper layer, and sterilize 0.15M
The mixture was diluted to 3.2 ml with NaCl and centrifuged at 50,000 rpm for 30 minutes. The precipitate was suspended by adding 0.15 M NaCl to 2.5 ml.
After the same operation was performed again, 3.2 ml of the glycolipid-containing liposome (particle diameter 0.17 μm) suspension of the present invention was obtained.

Comparative Example 1 A glycolipid-free liposome suspension 3.2 ml was obtained in the same manner as in Example 1 except that dipalmitoylphosphatidylglycerol (DPPG) 40 μmol was used instead of glycolipid A.

Example 1 (Measurement of ¹⁴ C and ³ H distribution rate in serum and tissue) Using the liposome suspensions obtained in Example 1 and Comparative Example 1, a test was performed according to the following procedure.

0.5 ml (per animal) of a liposome suspension was injected from the jugular vein of a rat. Blood sampling is 5, 15, 30, 60 minutes, 1, 2,
The test was performed from the femoral vein at 4, 6, and 20 hours. The collected blood was centrifuged at 2,000 rpm for 5 minutes, and 30 μl of the serum portion was collected.
1 ml of a tissue dissolving agent (Soren, Merk) was added, and the mixture was treated at 40 ° C. for 2 hours. Then, aqueous hydrogen peroxide and isopropyl alcohol were added to prepare a sample for measurement. Twenty hours later, the liver was excised from the rat, and a part thereof was treated in the same manner as the serum to obtain a measurement sample.

For the sample prepared above, ¹⁴ C and ³ H were measured by a liquid scintillation counter, and the blood concentration of the liposome at each time (the concentration at the time of injection) was determined from the obtained values.
100) and the distribution ratio (%) to the liver after 22 hours. The results are shown in Tables 1 and 2. Each value is an average of values obtained from five rats.

From Table 1, the liposome of Example 1 (the product of the present invention)
It can be seen that the liposome is present in blood at a higher concentration for a longer time than the liposome of Comparative Example 1 (comparative product). This becomes clearer by making a graph. That is,
According to FIG. 1 showing the change in the blood concentration of ³ H inulin over time, the blood concentration of the product of the present invention after 8 hours is 1.4 times that of the comparative product and 3 times after 20 hours.

In Table 2, the distribution of ¹⁴ C and ³ H to the liver was considerably lower than that of the comparative product. Therefore, the product of the present invention showed more reticuloendothelial tissue (liver) than the comparative product. It turns out that it is extremely difficult to be taken in.

The above results indicate that the liposome of the present invention efficiently avoids uptake by reticuloendothelial tissue and maintains a high blood concentration for a long time.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the time course of the blood concentration of ³ H inulin.

Claims (7)

(57) [Claims] 1. The method of claim 1, wherein uronic acid, uronic acid oligosaccharide, oligosaccharide composed of glucosamine and glucuronic acid, oligosaccharide composed of galactosamine and glucuronic acid, oligosaccharide composed of glucuronic acid and xylose, glucuronic acid and glucose are formed on the lipid membrane of the liposome. Oligosaccharide consisting of glucuronic acid and galactose, oligosaccharide consisting of glucuronic acid and mannose, oligosaccharide consisting of galacturonic acid and rhamnose, ketoaldonic acid, 2-acetamido-2-deoxyuronic acid, 2-acetamido-2 -Deoxyuronic acid oligosaccharide, 6-O-carboxyethyl-β
-D-glucose, 6-O-carboxymethyl-β-D
-Glucose, 6'-O-carboxymethyl-β-D-
Maltose and 6'-O-carboxyethyl-β-D-
A liposome containing a glycolipid comprising at least one kind of sugar and lipid selected from the group consisting of maltose and an O-glycoside bond or an S-glycoside bond. 2. The saccharide comprises uronic acid, uronic acid oligosaccharide, oligosaccharide comprising glucosamine and glucuronic acid, oligosaccharide comprising galactosamine and glucuronic acid, oligosaccharide comprising glucuronic acid and galactose, and glucuronic acid and mannose. The liposome according to claim 1, which is at least one member selected from the group consisting of oligosaccharides. 3. The liposome according to claim 1, wherein the saccharide is at least one selected from the group consisting of uronic acid, uronic acid oligosaccharide, oligosaccharide composed of glucosamine and glucuronic acid, and oligosaccharide composed of galactosamine and glucuronic acid. 4. The liposome according to claim 1, wherein the sugar is uronic acid and / or uronic acid oligosaccharide. 5. A lipid which forms a glycolipid by an O-glycosidic bond or an S-glycosidic bond with a sugar, wherein the lipid is a diacylglyceride, a dialkylglyceride, a sphingosine,
The liposome according to claim 4, which is at least one selected from ceramide, hydrocarbon, and cholesterol. 6. The liposome according to claim 1, wherein the glycolipid is contained in the lipid membrane in an amount of 1 to 50 mol%. 7. The liposome according to claim 1, wherein the glycolipid is contained in the lipid membrane in an amount of 5 to 30 mol%.