JPH08151334A - Liposome and preparation thereof - Google Patents

Abstract

PURPOSE: To provide liposome which comprises phosphatidyl choline containing docosahexaenoic acid in a high concentration as a membrane-forming lipid, thus is useful in controlled release of medicines with high stability in living bodies. CONSTITUTION: A phosphatidyl choline containing fatty acid composition more than 10%, preferably more than 20% of which are occupied by docosahexaenoic acid is used as a film-forming lipid to give this liposome preparation in a usual manner. The phosphatidyl choline is isolated by solvent fraction of cuttlefish skin followed by a combination of existing adsorption chromatography and ion-exchange chromatography. This liposome can be included in medicines and examination reagents for human and animals, for example, ascorbic docosahexaenoate (DHA-AS) has both antioxidant action of AS and vitamin C effect, as well as the physiological activity of high-degree unsaturated fatty acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention contains a liposome containing phosphatidylcholine as a membrane-forming lipid, in which docosahexaenoic acid accounts for 10% or more of the constituent fatty acid composition, and ascorbic acid ester of docosahexaenoic acid, which are useful for controlling drug release and the like. Regarding the formulation.

[0002]

2. Description of the Related Art Liposomes are vesicles formed from lipid artificial membranes, have a high affinity for living organisms, are made of biodegradable materials, and can hold various substances. Therefore, it has been investigated to incorporate a drug or the like into various vesicles in the form of various microcapsules and use the drug in a drug release control or drug delivery system.

To this end, liposomes are known to
Stable in vivo until at least releasing the drug
However, it is required to release the encapsulated drug at a constant rate.
It One of the factors that destroy liposomes in vivo
As one, phospholipase A ₂(PLA₂) Is known
Have been.

Conventionally, as the phospholipid as the liposome-forming lipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk phosphatidylcholine, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine and the like have been used. It is said that the acyl group of the lipid is preferably a saturated acyl group (for example, JP-A-6-9).
No. 374, etc.), the use of liposomes containing phosphatidylcholine containing a large amount of docosahexaenoic acid, which is a highly unsaturated fatty acid, as a membrane-forming lipid is not known at all. This docosahexaenoic acid (DHA) is known to have a learning ability-improving action, a lightness-discriminating ability-improving action, and an antiallergic action, which are not found in the same n-3 series icosapentaenoic acid (EPA) or α-linolenic acid. Therefore, it is expected to be applied to medicines, health foods, and cosmetics in the modern society where senile dementia and adult diseases are becoming serious.

[0005]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a liposome which is highly stable in vivo and useful for controlling drug release and the like, and a preparation containing the ascorbic acid ester of docosahexaenoic acid. To do.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that when phosphatidylcholine, in which docosahexaenoic acid accounts for 10% or more of the constituent fatty acid composition, is used as a membrane-forming lipid, It was found that the liposome can be easily formed by the method and that it has excellent stability. Furthermore, they have found that the affinity of the ascorbic acid ester of docosahexaenoic acid for the hydrophobic portion rich in docosahexaenoic acid is high, and that an excellent liposome preparation can be formed, and the present invention has been completed.

That is, the present invention relates to a liposome containing phosphatidylcholine as a membrane-forming lipid in which docosahexaenoic acid accounts for 10% or more of the constituent fatty acid composition, and a preparation thereof containing an ascorbic acid ester of docosahexaenoic acid.

The phospholipid as the membrane-forming lipid of the liposome of the present invention is composed of docosahexaenoic acid as a constituent fatty acid composition.
Any phosphatidylcholine may be used as long as it accounts for 0% or more, but docosahexaenoic acid is 2% from the viewpoint of film stability.
It is more preferably at least 5%, and for example, synthetically produced phosphatidylcholine or phosphatidylcholine (sPC) separated from squid skin is preferable. Squid skin contains about 2% of phospholipids per wet weight, about 50% of the phospholipids are choline-type phospholipids, and about 20% are ethanolamine-type phospholipids. Docosahexaenoic acid accounts for about 50% of this. The PC bond type may be either a diacyl type or an alkylacyl type, but it is preferable that a large amount of diacyl type is contained.

The morphology of the membrane may be either multilamellar vesicles (MLV) or unilamellar vesicles, and small unilamellar vesicles (SUV) or large unilamellar vesicles (L).
UV).

Examples of the squid used as a raw material include purple squid, american squid, squid, cuttlefish and firefly squid. Squid skin is present in large quantities as waste after removing the edible portion, and is available virtually free of charge.
Phosphatidylcholine can be easily isolated from other phospholipids separately from other phospholipids by solvent separation from squid skin by a conventional method and then by combining existing adsorption chromatography and ion exchange chromatography.

That is, the extraction of the lipid component is preferably carried out by freeze-drying the squid skin before the extraction of the lipid component and then crushing the squid finely to expand the surface area. Usually, it is suitably achieved by using an organic solvent. It As the organic solvent used, it is possible to use an ordinary solvent that dissolves phospholipids alone or in combination, for example, chloroform, ether, butyl alcohol, chloroform-methanol system,
n-hexane-ethanol aqueous system, chloroform-n-hexane system, chloroform-ether system, chloroform-
Examples include ethanol type. Of these solvents,
Chloroform-methanol system is preferable, and particularly, the ratio of chloroform is preferably 50% or more, more preferably 65% or more from the viewpoint of good extraction efficiency of lipid components.

Then, the obtained lipid component is fractionated to obtain a phospholipid containing DHA. The fraction is
Generally, after removing the organic solvent, it can be carried out by any of the methods usually used for fractionating phospholipids from lipid components. However, since a large amount can be processed, a fractionating solvent is added to separate insoluble substances. It is preferable to carry out the above method, that is, the solvent fractionation method. As a solvent for separation,
Usually, an organic solvent used for fractionating lipids can be used, and examples thereof include acetone, acetic acid, chloroform and ether. Of these solvents, acetone is particularly preferable because of its high fractionation efficiency. Further, magnesium chloride, calcium chloride or the like may be added to the solvent and the fractionation may be performed in the coexistence thereof, and heating, cooling or the like may be performed as necessary. Further, the obtained fraction is re-fractionated by column chromatography and the desired phospholipid fraction is collected, whereby a highly pure phospholipid composition can be obtained in a high yield. As column chromatography, silica-based chromatography, hydrophobic chromatography, ion exchange chromatography and the like can be used. Further, in order to fractionate phosphatidylcholine from phosphatidylethanolamine (PE), the phospholipid composition obtained by solvent fractionation is first subjected to silica-based chromatography to separate PE,
Phosphatidylcholine can be separated by subjecting it to ion exchange chromatography.

Each of the phospholipids thus obtained can be analyzed by thin-layer chromatography and NMR to identify its type. Furthermore, saponification, methyl esterification, or methanolysis can be performed by a conventional method. According to the method, after the fatty acid methyl ester is obtained, it can be analyzed by gas chromatography to determine the constituent fatty acid composition.

Further, these phospholipids are treated with phospholipase A ₂ , and the produced free fatty acids and lysophospholipids are fractionated by thin layer chromatography, and then fatty acid analysis is carried out as described above, whereby fatty acid The phospholipid intramolecular distribution can be determined.

The constituent fatty acids of sPC isolated from squid skin obtained by the above-described method were DHA, EPA, stearic acid, palmitic acid, etc., and the DHA content was generally as high as about 50%.

The liposome of the present invention and its preparation are prepared by using the phosphatidylcholine isolated as described above.
It can be prepared by a conventional method.

As the constituent component of the membrane lipid, cholesterol and / or a charged lipid may be added in addition to the above phosphatidylcholine to form a liposome preparation. Examples of the charged lipid include PE, phosphatidylglycerol, phosphatidylserine and phosphatidyl. Inositol, phosphatidic acid, dicetyl phosphoric acid, stearylamine, fatty acids and the like may be added. Also, as a membrane stabilizer, a cryoprotectant, an antioxidant, cholesterol,
It is also possible to add sterols such as cholestanol, sugars, glycolipids, glycerin, polyethylene glycol, tocopherols and ascorbic acid. The addition amount of these is preferably 1 mol or less with respect to 1 mol of phosphatidylcholine, and one example thereof is that the above-mentioned phosphatidylcholine is first dissolved in a solvent together with appropriate amounts of cholesterol and phosphatidic acid, and the solvent is placed in a container such as a flask. Remove and apply evenly to the inner wall of the container. MLV-type liposomes can be obtained by adding distilled water to the dried product and suspending it. In addition, LUV-type liposomes can be obtained by freeze-drying a dried product prepared in the same manner and suspending the freeze-dried product in a phosphate buffer (PBS).
By ultrasonically crushing this, SUV type liposomes can be obtained.

The liposome of the present invention can contain various drugs such as human and veterinary drugs and test agents according to a conventional method, and a liposome preparation containing these drugs is also within the scope of the present invention. Is. In particular, since it is fat-soluble, it is difficult to dissolve in water and it is necessary to use an organic solvent or a solubilizing agent. Further, oral administration is likely to be decomposed in the digestive organs, and in many cases blood administration is preferable. In the form, it is required to ensure stability in blood. For example, ascorbic acid ester of docosahexaenoic acid, ascorbic acid, antioxidative action and vitamin C effect and highly unsaturated fatty acid bioactive effect. Is a highly applicable ascorbic acid ester (Japanese Patent Application Laid-Open No. 3-99073), which is a cardiovascular drug, especially angina,
Calcium antagonist effective for prevention and treatment of ischemic heart disease such as myocardial infarction and hypertension (WO94 / 200
No. 92).

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[0020]

【Example】

Reference Example 1: Isolation and analysis of phospholipids contained in squid skins Wet squid skins with a wet weight of 10 g were lyophilized to 1.6
g of dried product was obtained. After crushing this finely, it was extracted with a mixed solvent of chloroform: methanol = 2: 1 to give 0.26
g lipid component was obtained. 40 ml of acetone for this lipid component
Was added, and the mixture was stirred while being cooled, and neutral lipids were separated and filtered to obtain 0.22 g of acetone-insoluble matter. This was subjected to silica gel column chromatography to develop 500 ml, 400 ml, and 2000 ml in the order of chloroform: methanol = 85: 15, 80:20, 75:25.
This allowed fractionation into PE and other phospholipid mixtures. Next, the other phospholipid mixture was subjected to Sepharose column chromatography, and chloroform: methanol = 1: 4, acetic acid, chloroform: methanol =
100 ml of 1: 4 + ammonium acetate (0.2%) was sequentially developed. This allows s from other phospholipid mixtures
PC could be isolated. The isolated sPC and PE were each 0.
It was 11 g and 0.04 g. These phospholipids are NM
After confirming each structure with R, the fatty acid composition was analyzed. As a result, DHA, EPA, stearic acid, and palmitic acid were 51%, 5%, 2%, 33% for sPC, 19%, 33% for PE, respectively. It was 12% and 6%. In addition, when the intramolecular distribution of these fatty acids was analyzed, sn-
Second place was all DHA. The sn-2 position of PE is DH
A or EPA.

Reference Example 2: Isolation and analysis of phospholipids contained in squid skins The skins of squid of 30 g wet weight were freeze-dried and 4.8.
g dry matter was obtained. After crushing this finely, it was extracted with a mixed solvent of chloroform: methanol = 2: 1 to give 0.61
g lipid component was obtained. 40 ml of acetone for this lipid component
Was added, and the mixture was stirred with cooling, and neutral lipids were separated and filtered to obtain 0.54 g of acetone-insoluble matter. This was subjected to silica gel column chromatography to develop 500 ml, 400 ml, and 2000 ml in the order of chloroform: methanol = 85: 15, 80:20, 75:25.
This allowed fractionation into PE and other phospholipid mixtures. Next, the other phospholipid mixture was subjected to Sepharose column chromatography, and chloroform: methanol = 1: 4, acetic acid, chloroform: methanol =
100 ml of 1: 4 + ammonium acetate (0.2%) was sequentially developed. This allowed sPC to be isolated from other phospholipid mixtures. The isolated sPC and PE were 0.29 g and 0.15 g, respectively. After the respective structures of these phospholipids were confirmed by NMR, the fatty acid composition was analyzed. As a result, DHA, EPA, stearic acid, and palmitic acid were found to have 49%, 6%, 2%, 33% PE in sPC, respectively.
Was 18%, 33%, 11% and 6%. In addition, when the intramolecular distribution of these fatty acids was analyzed, the sPC
All n-2 positions were DHA. The sn-2 position of PE was DHA or EPA.

Example 1 Preparation of Liposomes The sPC isolated in Reference Example 1, cholesterol (chol), and dipalmitoylphosphatidic acid (DPPA) were mixed in 8:
It was dissolved in a mixed solvent of chloroform / methanol = 2/1 at a molar ratio of 5: 1. This solution was placed in a pear-shaped flask, and the solvent was distilled off using a rotary evaporator. To ensure that the lipid membrane is uniformly formed on the inner wall of the flask,
If necessary, 0.2 ml of chloroform was added, and the solvent was distilled off again with an evaporator. 0.5 ~ with a vacuum pump
After being kept under reduced pressure for 1 hour, it was suspended in about 0.6 ml of distilled water, transferred to a screw cap test tube and freeze-dried. PBS ^- (0.8%
NaCl, 0.02% KCl, 0.115% Na ₂ HPO ₄ , 0.02% KH ₂ PO ₄ ) 2
It was suspended in ml, 0.5 ml of 0.1 M CaCl ₂ was added to divide into two, and one was subjected to ultrasonic disruption. As a result of observation using a 400 × optical microscope, the sample subjected to ultrasonic crushing was found to have liposomes with a particle size of 1 μm or less (hereinafter, sP
C / SUV), and the sample which was not crushed was a liposome having a particle size of about 1 μm (hereinafter, sPC).
/ LUV).

Reference Example 3: Preparation of liposomes Substituting soybean phosphatidylcholine (soybean PC) for sPC, the molar ratio was soybean PC / chol / DPPA = 4: 5:
1, the same operation as in Example 1 was carried out, and soybean PC-derived liposomes (hereinafter, soybean PC / LUV and soybean P
C / SUV) was prepared.

Test Example 1 0.2 ml of each sPC / LUV, sPC / SUV, soybean PC / LUV and soybean PC / SUV liposome solution.
Each was dispensed into four screw cap test tubes, and one was stored in ice as a sample for 0 hours of reaction. PL for porcine pancreas in the remaining 3
Add 10 μl of A ₂ (11 units),
After incubating for 0, 60 minutes, 10 mM
The reaction was stopped by adding 30 μl of EGTA. Lipids were extracted from each sample according to the method of Forti and subjected to silica gel thin layer chromatography (chloroform / methanol /
Water = 65/25/2). The free acid fraction was scraped off, converted into fatty acid methyl ester by the hydrochloric acid-methanol method, and quantified by gas chromatography. sPC / LU
For V and sPC / SUV, docosahexaenoic acid (2
2: 6 (n-3)), for soybean PC / LUV and soybean PC / SUV, for linoleic acid (18: 2 (n-6)), the corresponding number of moles (nmol) of free acid in phosphatidylcholine. The ratio (hydrolysis rate,%) to the number of moles of the acid used was calculated and compared. The results are shown in FIGS. 1 and 2.

From these results, the liposome prepared from the phosphatidylcholine containing a large amount of docosahexaenoic acid of the present invention has a smaller amount of free acid than the liposome containing a general soybean PC, and is less likely to be decomposed by the enzyme. It is apparent that it has excellent stability.

Example 2 Preparation of Liposome Containing Ascorbic Acid Ester of Docosahexaenoic Acid Using Phosphatidylcholine (sPC) Obtained from Squid Skin 5 μmol of sPC isolated in Reference Example 1, ascorbic acid ester of docosahexaenoic acid ( DHA-As) 4 μmo
l, DPPA 0.5 μmol, Cholesterol 2.5 μmol are dissolved in chloroform and transferred to a pear-shaped flask, and the solvent is distilled off using a rotary evaporator so that a lipid membrane is uniformly formed in the flask, and then a vacuum pump And further dried under reduced pressure. Next, 0.2 ml of distilled water was added to the inside of the flask to suspend this thin film. The particle size of the obtained MLV-type liposome was observed using an optical microscope with a magnification of 400, and it was confirmed that the particle size was 1 μm or less.

Reference Example 4: Preparation of liposome containing ascorbic acid ester of docosahexaenoic acid using egg yolk PC (ePC) 5 μmol of ePC, 4 μmol of DHA-As, DPP
A 0.5 μmol and cholesterol 2.5 μmol were dissolved in chloroform, and M was dissolved in the same manner as in Example 1.
LV (DHA-As / ePC) was obtained.

Reference Example 5: Dipalmitoyl PC (DPP
Preparation of liposome containing ascorbic acid ester of docosahexaenoic acid using C) DPPC 5 μmol, DHA-As 4 μmol, DPP
A 0.5 μmol and cholesterol 2.5 μmol were dissolved in chloroform, and the same procedure as in Example 1 was repeated.
MLV (DHA-As / DPPC) was obtained.

[0029] Test Example 2: Stability embodiment of liposomes containing ascorbic acid ester of docosahexaenoic acid 2, the liposome solution 0.2ml of MLV obtained in Reference Example ^{4,5 PBS - (0.8% Na Cl} , 0.02% KCl, 0.115%
0.8 ml of NaHPO ₄ , 0.02% KH ₂ PO ₄ ) was added and suspended, and 0.2 ml of each liposome solution was dispensed into three test tubes with a screw, one of which was used as a sample for 0 hour of reaction. The other two were samples left after being left for 12 and 24 hours at 37 ° C. This sample was centrifuged (14,000 rpm (about 24,000 xg)) to separate liposomes. The liposomes were washed twice with PBS ^- and then centrifuged to extract lipids according to the method of Forti. This lipid was applied to silica gel TLC, phosphatidylcholine and DHA-As were scraped off, and each constituent fatty acid methyl ester was prepared by the hydrochloric acid-methanol method to prepare GL.
C analysis was performed to examine the fatty acid composition. The results are shown in FIGS.
Summarized in 5.

In the case of using sPC, as is clear from FIG. 3, the amount of MLV produced was smaller than in the case of using ePC or DPPC, but the change in PC amount with time was the smallest and stable. And, even if the amount of DHA-As contained in MLV varies, MLV prepared from sPC has ePC or DPPC.
More stably held DHA-As (Fig. 4).
Furthermore, the MLV of this sPC contained a large amount of DHA-As (FIG. 5).

[0031]

INDUSTRIAL APPLICABILITY The liposome of the present invention has high in vivo stability by using phosphatidylcholine containing DHA at a high concentration as a membrane-forming lipid.
The preparation is, for example, DHA- which has a useful physiological action.
As can be contained at a high concentration, and it becomes possible to control the release of DHA-As in the living body.
It can be expected that the physiological activity of A-As is effectively acted on.

[Brief description of drawings]

FIG. 1 is a graph showing the stability test results for sPC / LUV obtained in Example 1 and soybean PC / LUV obtained in Reference Example 3 against PLA ₂ .

FIG. 2 is a graph showing the stability test results for PLA ₂ of sPC / SUV obtained in Example 1 and soybean PC / SUV obtained in Reference Example 3.

FIG. 3 is a 37 ° C. MLV containing DHA-As made from the various PCs obtained in Example 1, Reference Example 4 and Reference Example 5;
The change with time is shown by the amount of PC.

FIG. 4 shows the changes over time of MLVs made from the various PCs obtained in Example 1, Reference Example 4 and Reference Example 5 at 37 ° C. in terms of the amount of DHA-As.

FIG. 5 shows changes with time of MLVs made from various PCs obtained in Example 1, Reference Example 4 and Reference Example 5 at 37 ° C. in DHA-As / PC ratio.

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[Procedure amendment]

[Submission date] August 15, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

Example 1 Preparation of Liposomes The sPC isolated in Reference Example 1, cholesterol (chol), and dipalmitoylphosphatidic acid (DPPA) were mixed in 8:
It was dissolved in a mixed solvent of chloroform / methanol = 2/1 at a molar ratio of 5: 1. This solution was placed in a pear-shaped flask, and the solvent was distilled off using a rotary evaporator. To ensure that the lipid membrane is uniformly formed on the inner wall of the flask,
If necessary, 0.2 ml of chloroform was added, and the solvent was distilled off again with an evaporator. 0.5 ~ with a vacuum pump
After being kept under reduced pressure for 1 hour, it was suspended in about 0.6 ml of distilled water, transferred to a screw cap test tube and freeze-dried. PBS ^- (0.8%
NaCl, 0.02% KCl, 0.115% Na ₂ HPO ₄ , 0.02% KH ₂ PO ₄ ) 2
It was suspended in ml, 0.5 ml of 0.1 M CaCl ₂ was added to divide into two, and one was subjected to ultrasonic disruption . The sample was subjected to ultrasonic crushing, s PC / SU V, the sample was not carried out a fracture you abbreviated as s PC / LUV.

Claims (3)

[Claims] 1. A liposome containing phosphatidylcholine, in which docosahexaenoic acid accounts for 10% or more of the constituent fatty acid composition, as a membrane-forming lipid. 2. The liposome according to claim 1, wherein the phosphatidylcholine is phosphatidylcholine isolated from squid skin. 3. The liposome preparation according to claim 1, which contains an ascorbic acid ester of docosahexaenoic acid.