WO2003015753A1

WO2003015753A1 - Liposome preparations

Info

Publication number: WO2003015753A1
Application number: PCT/JP2002/008380
Authority: WO
Inventors: Yoshitaka Ogata; Hiroshi Gotou
Original assignee: Terumo Kabushiki Kaisha
Priority date: 2001-08-20
Filing date: 2002-08-20
Publication date: 2003-02-27
Also published as: JPWO2003015753A1

Abstract

Liposome preparations comprising liposomes and a substance having been incorporated in the liposomes, wherein a film agent constituting the liposomes contains a phospholipid and at least one higher saturated fatty acid having 10 to 20 carbon atoms and the molar ratio of the higher saturated fatty acid to the phospholipid ranges from 30 mol% to 50 mol%. These preparations, in which a less expensive film agent is employed as a liposome material, are excellent in the drug retention ratio within the liposomes.

Description

Akira Liposomal formulation Technical field

The present invention relates to an improved drug-retaining ribosome preparation, and more particularly, to an improved drug-retaining ribosome preparation in which various bioactive substances are encapsulated inside ribosomes (vesicles). Background art

Ribosomes are defined as closed vesicles with a membrane composed primarily of lipids. Ribosomes, which are the endoplasmic reticulum of the lipid bilayer membrane, can be used as a drug delivery system because they can encapsulate various substances inside and have excellent biocompatibility because they are formed from biological substances. Ribosomes have been used in many studies as models of biological membranes, but have also been applied to drug delivery systems (DDS). It is also used in the field of genetic engineering as a carrier for genes and antisense. By the way, drug encapsulation changes the biodistribution of the drug and the residence time in the bloodstream. It will also improve accessibility to target organs, reduce side effects of the drug, and allow for sustained release. The advantages of using ribosomes include low toxicity and antigenicity, and metabolism in vivo because the phospholipid used is a biological component. In addition, the size and lipid composition of ribosomes can be easily adjusted, and many things such as water-soluble drugs, fat-soluble drugs, polymers, and proteins can be encapsulated. Surface modification with quality, antibodies, lectins, etc. is also easy. In addition, by imparting functions such as membrane fusibility and control of adhesion to specific cells to ribosomes, drug capsules for the purpose can be constructed.

In this way, drug-retained liposomal preparations can be used to stabilize drugs that are unstable in the living body or to gradually release drugs in the living body, and selectively transfer drugs to specific organs. Use is also considered as a means for this. For example, ribosome preparations containing hemoglobin, insulin, heparin, peroxidase, and various other anticancer and antifungal agents are known as preparations for stabilization and sustained release. As a preparation for the purpose of rapidly transferring a drug to ribosomes, a ribosome preparation containing ubide force renone, cytosine arabinoside, steroids, and other various anticancer and antifungal agents is known.

Phospholipids used as membranes in conventional drug-retaining liposomal preparations include unsaturated lecithin such as egg yolk lecithin or soy lecithin, purified saturated lecithin hydrogenated to these, phosphatidylcholine further separated and purified from these, and phosphatidyl Examples include purified lecithin such as ethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin, and synthetic lecithin such as dimyristoyl lecithin, dipalmityl lecithin, and distearoyl lecithin. These have good affinity to the living body and are used safely in parenteral preparations such as injections. However, conventionally used ribosome membrane materials do not always have a sufficient drug retention. That is, the amount of the drug contained in the unit ribosome drug is not sufficient, and a more efficient membrane material is demanded. Furthermore, conventional ribosome membrane materials made from unsaturated lecithin and unsaturated fatty acids (Japanese Patent Application Laid-Open No. 60-155109) are stable. The membranes were inadequate, and the membrane was relatively easily destroyed in vitro or in vivo, resulting in short life in vivo and release of drugs. A ribosome preparation using purified lecithin and synthetic lecithin with higher stability is very expensive, and there is a limit in using purified lecithin and synthetic lecithin as a raw material for the preparation. Further, in the range of a conventionally known method for producing a ribosome containing 5 to 15 watts of an unsaturated higher fatty acid having 18 to 20 carbon atoms (Japanese Patent Application Laid-Open No. 60-155109), when a saturated fatty acid is used, However, there was a problem that the drug retention of liposomes was significantly reduced.

Therefore, an object of the present invention is, firstly, to provide a drug-retaining liposome preparation having an excellent drug retention rate in the ribosome.

A second object of the present invention is to provide an inexpensive membrane agent as a liposome drug material. Disclosure of the invention

That is, the present invention provides the following inventions.

(1) In a liposomal preparation comprising a liposome and a substance incorporated in the liposome, the membrane agent constituting the ribosome contains a phospholipid and at least one kind of a higher saturated fatty acid having 10 to 20 carbon atoms. A ribosome preparation, wherein the content of the higher saturated fatty acid is 30 raol% to 50 mol% in a molar ratio in the ribosome component.

(2) The liposome preparation according to (1), wherein the phospholipid is a saturated phospholipid or a hydrogenated phospholipid.

(3) The method according to (1) or (2), further comprising cholesterol as the film agent. Ribosome preparation.

(4) An aggregation inhibitor having a hydrophilic polymer chain portion and a hydrophobic portion is further bound to the ribosome surface, and the aggregation inhibitor has the hydrophobic portion immobilized on the lipid layer in the liposome. The ribosome preparation according to any one of (1) to (3), wherein the hydrophilic polymer chain part extends outward from the liposome surface.

(5) The liposome preparation according to any one of (1) to (4), wherein the substance incorporated into the ribosome is a physiologically active substance.

(6) The liposomal preparation according to (5), wherein the physiologically active substance is hemoglobin. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the fatty acid charge ratio and the protein yield.

Figure 2 is a graph showing the fatty acid charge ratio and the amount of force-pressed protein per lipid.

Figure 3 is a graph showing fatty acid loading ratio and ribosome stability (protein leakage rate).

FIG. 4 is a graph showing the carbon number of the fatty acid carbon chain, the protein yield of the liposomal preparation, and the protein amount per lipid amount. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

As a membrane agent used in the drug-retaining ribosome preparation of the present invention, a lipid suitable for preparing a liposome is used. Soy lecithin and yolk lecithin as phospholipids Any phospholipids such as natural unsaturated phospholipids, synthetic phospholipids, and natural hydrogenated phospholipids obtained by hydrogenating natural unsaturated phospholipids can be used. Since all natural phospholipids contain unsaturated fatty acids, hydrogenated phospholipids or synthetic phospholipids in which unsaturated fatty acids of the above natural phospholipids are saturated with hydrogen are required to achieve the object of the present invention to a higher degree. It is more effective to use.

Representative examples of the phospholipid used in the present invention include lecithin, phosphatidylethanolamine, phosphatidylinosyl], phosphatidylserine, phosphatidylglycerol, sphingomyelin, and cardioribine. Further, there may be mentioned those obtained by hydrogenating them according to a conventional method. In particular, a hydrogenated natural lecithin obtained by hydrogenating soybean lecithin, egg yolk lecithin, corn lecithin, cottonseed oil lecithin, nayu lecithin and the like is preferably used.

As the higher saturated fatty acid in the present invention, a higher fatty acid having 10 to 20 carbon atoms is desirable, and examples thereof include ricopric acid, lauric acid, myristin shun, balmitic acid, stearic acid, and eicosanoic acid. . These higher saturated fatty acids can be used alone or as a mixture. Since the hydrophobic carbon chain of the phospholipid suitably used as the ribosome membrane constituent lipid has about 14 to 18 carbon atoms, the higher saturated fatty acid having 14 to 18 carbon atoms can be obtained from the affinity with the phospholipid. More desirable. The molar ratio of the higher saturated fatty acid to the liposome component should be 30 mol% or more, and the molar ratio does not cause micelle formation with the phospholipid. Even when the blending amount of the higher saturated fatty acid is less than 30 mol%, liposomes are formed and the drug has the ability to retain a drug, but in order to improve the drug retention, the blending ratio of the higher saturated fatty acid is 30 mol1 in a molar ratio. This is necessary. Further, when the content is 40 mol% or more, the affinity and stability which are the object of the present invention It is more preferable because it exhibits excellent properties as a drug, a drug retention rate, and an inexpensive film agent. Phospholipids cannot retain drugs as ribosomes unless they are used in a concentration range that results in a lamellar structure.However, if the amount of higher saturated finger acid is excessively increased, micelles formed by higher saturated fatty acid inhibitors are formed. Phospholipids are taken up and do not form liposomes. At this time, the blending ratio of the higher saturated fatty acid varies depending on the number of carbon atoms and conditions of the fatty acid, but it is over about 50 mol% in molar ratio. Therefore, higher fatty acids will not form ribosomes unless used at a ratio below this ratio, and the drug retention efficiency will be extremely low or impossible. Preferably, the molar ratio is 45 mol% or less. Therefore, the molar ratio of the higher saturated fatty acid to the phospholipid is 30 mol to 50 mol%, preferably 40 mol% to 45 mol%.

A sterol such as cholesterol or tocopherol can be added to the membrane material of the present invention in order to increase the strength of the membrane. In addition, a substance for controlling transfer to a target organ in a living body and sustained release properties can be used. It is also possible to add.

The retained substance incorporated and retained in the ribosome preparation of the present invention is not particularly limited as long as it does not inhibit ribosome formation, but it is unstable in vitro or in vivo, and in the bloodstream. A physiologically active substance which is desired to be stably retained in the stomach or a substance which is desired to be rapidly distributed to a specific organ is preferably used. Examples of such substances include hemoglobin, insulin, heparin, perokinase, shrimp dicarenone, methotrexate neomycin, prepomycin, tetracycline, cytochrome C, asparaginase, and cytosine arabinoside. . Other than drugs, especially markers, antisense, plasmid ゃ DM, RNA, etc., which are effective when administered in vivo, etc. There is no restriction. The drug-carrying ribosome preparation of the present invention is produced by a method known per se. For example, natural phospholipids, hydrogenated natural phospholipids, at least one of the above-mentioned higher saturated fatty acids and, if desired, sterol are dissolved in a suitable solvent such as chloroform or ethanol, and the solvent is distilled off from the resulting solution. To prepare a lipid mixture. An aqueous solution of a drug such as a physiologically active substance is added to the obtained lipid mixture, and the obtained mixture is vigorously shaken, stirred or sonicated to uniformly disperse the aqueous drug solution. The drug not taken into the ribosome is removed from the dispersion to obtain a drug-retaining ribosome preparation. The liposomal preparation thus obtained is prepared, if necessary, as a suspension preparation dispersed in a physiologically acceptable aqueous solution, for example, physiological saline. The liposome preparation of the present invention is administered as a parenteral preparation such as an injection. The ribosome of the present invention may be freeze-dried under ordinary conditions.For example, it is preferable to freeze at −20 ° C. to −80 ° C. and sublimate the ice under a reduced pressure of 0.3 torr or less. No. In order to form a better freeze-dried cake, commonly used excipients such as mannitol, dextrin, glycine and the like may be added. In the present invention, a ribosome preparation is defined as a preparation in which various substances to be retained are contained inside the ribosome.

The ribosome preparation of the present invention may further contain an aggregation inhibitor having a hydrophilic polymer chain portion and a hydrophobic portion. The amount of the aggregation inhibitor can be preferably 0.01 to 5% by mass, more preferably 0.01 to 1% by mass, based on the liposomal preparation. When the ribosome is modified with “hydrophilic polymer-bound lipid”, if the substance to be encapsulated can be heated, it can be modified to about 5% by mass. If it is less than 0.01% by mass, the effect of suppressing aggregation is hardly obtained even if it is modified. Also, if it is 1% by mass or less It can be modified without heating and can contain heat-labile proteins such as bioactive substances and hemoglobin, and has a sufficient aggregation-suppressing effect. When an aggregation inhibitor is added to the liposome preparation, the hydrophobic part is stably inserted into the ribosome surface and fixed to the lipid layer in the liposome, and the hydrophilic polymer chains move outward from the liposome surface. It has the function of elongating and suppressing ribosome aggregation.

Examples of the "aggregation inhibitor" include, as the hydrophobic part, various saturated 'unsaturated fatty acids, sterols, polyoxypropylene alkyl or glycerin fatty acid esters, and phospholipids, and phospholipids are preferable.

The hydrophilic polymer chains include polyalkylene glycol, polyvinyl alcohol, alternating copolymer of styrene and maleic anhydride, alternating copolymer of divinyl ether and maleic anhydride, polyglycolic acid, polylactic acid, and dextran and pullula. And polysaccharides such as ficoll, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, and carrageenan. Among them, polyethylene glycol is most desirable because of its remarkable effect of improving blood retention. Polyethylene glycol-linked phospholipids and polyethylene glycol-linked cholesterol are preferred.

Hereinafter, the present invention will be described more specifically with reference to examples. However, these are only examples of the present invention, and the present invention is not limited thereto.

(Example 1)

A mixture of hydrogenated soybean lecithin (HSPC: molecular weight 790) as phosphatide, cholesterol (molecular weight 376) and stearic acid (molecular weight 278) as higher fatty acid. Different mixed lipids (Table 1) were prepared by dissolving in 10 ml of t-BuOH and freeze-drying to remove t-BuOH. Hemoglobin (45wt%) solution, 20ml was added to this and a high-speed stirrer

(Cleamix, CLM-0.8S) and emulsified (15,000 rpm, lmin, 3 times at 20 ° C or less). After emulsification, 30 ml of physiological saline was added to each treated material, and the unencapsulated hemoglobin was centrifugally washed three times at 40,000 G and 60 min. After that, the mixture was dispersed in 20 ml of physiological saline and filtered using a 0.45 m filter, and a polyethylene glycol (PEG) -bound phospholipid was added as an anticoagulant to a concentration of 0.1% by mass. A decorated ribosome-encapsulated hemoglobin agent was obtained.

Table 1 Preparation of mixed lipids]

Samp 1 e

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Fatty acid

mol ° / o 0.00 6.7 12.5 22.2 26.3 30.0 33.3 36.3 39.1 41.2 44.0 46.2 50.0 60.0 66.7

HSPC

(g) 0.67 0.64 0.62 0.58 0.57 0.55 0.54 0.52 0.51 0.49 0.48 0.47 0.45 0.38 0.34 Cholester. -Le

(g) 0.33 0.32 0.31 0.29 0.28 0.28 0.27 0.26 0.25 0.25 0.24 0.23 0.22 0.19 0.17 Stearic acid

(g) 0.00 0.03 0.07 0.12 0.15 0.17 0.20 0.22 0.24 0.26 0.28 0.30 0.33 0.42 0.50

The concentration of hemoglobin in the liposomal encapsulated hemoglobulin preparations of each formulation ratio obtained in this way was measured by atomic absorption, and the concentrations of HSPC, cholesterol, and stearic acid were measured by high-performance liquid chromatography. did.

From these results, the yield of protein, which is hemoglobin encapsulated in liposomes, was determined by the following equation.

"Protein yield (%) = (Amount of protein retained in ribosome Z Amount of protein input at adjustment) X 100

As a result, when the molar ratio of the fatty acid to the phospholipid was less than 30 mol, the protein yield was about 10% or less, but when the molar ratio was in the range of 30 mol% to 50 mol%, the protein yield was 12% or more. It was found that at a molar ratio of about 40 mol%, the protein yield increased to 16% (Fig. 1).

Considering efficient administration of a drug encapsulated in ribosomes to a living body, the larger the amount of encapsulated protein per amount of lipid, the better.Therefore, the amount of encapsulated protein per amount of lipid is calculated by the following formula. I asked.

"Encapsulated protein amount per lipid amount == (protein retention amount in ribosome (HS PC amount + cholesterol amount + stearic acid amount))"

From these results, when the molar ratio of fatty acid to phospholipid is less than 26 mol%, except for the case where no fatty acid is contained (in this case, almost no ribosome-encapsulated hemoglobin preparation was formed) Although the amount of encapsulated protein remained only about 1.2, the amount of encapsulated protein per lipid increased remarkably when the molar ratio was in the range of 26mol-50moi%, and the amount of lipid was increased at the molar ratio of about 46mol%. The force per unit The amount of protein in the form of peptide was increased to 1.7 (Fig. 2). In addition, in order to evaluate the stability of in-vitro and in-vivo drug leakage from the liposomal preparation, the ribosome-encapsulated hemoglobin preparation at each mixing ratio was placed in a 50-ml centrifuge tube, and at 37 After shaking 60 times per minute for 3 hours, the mixture was centrifuged at 40,000 G for 60 minutes, and the amount of hemoglobin leaked into the supernatant was measured. The value obtained by dividing the amount of leaked hemoglobin by the amount of hemoglobin in the original liposome-encapsulated hemoglobin preparation is shown in FIG. 3 as the protein leakage rate ().

As a result, the stability of the liposome formulation is improved from a molar ratio to phospholipid of about 12 mol%, and is stable up to a molar ratio of 50 mol%, and is particularly stable in a range of 33 mol% to 46 mol%. Do you get it. It was also found that when the molar ratio exceeds 5 Omol%, the stability of the liposome formulation is adversely affected.

(Example 2)

HSPC (molecular weight: 790), cholesterol (molecular weight: 376) as phospholipids, lauric acid (molecular weight: 200), myristic acid (molecular weight: 228), palmitic acid (molecular weight: 256), stearic acid (higher saturated fatty acids) 1 g of a mixed lipid containing 33.3 mol of a molecular weight of 278) in a molar ratio to HSPC was prepared in the same manner as in Example 1. A hemoglobin (45w) solution (20 ml) was added thereto, and the mixture was emulsified (15,000 rpm, lmin, 3 times at 20 ° C or lower) using a high-speed stirrer (CLM-0.8S). After emulsification, 30 ml of physiological saline was added to each treated material, and the unencapsulated hemoglobin was washed by centrifugation at 40,000 G for 60 minutes three times. After that, the mixture was dispersed in 20 ml of physiological saline and filtered using 0.45 45 Filtration. Polyethylene glycol-linked phospholipid was added as an anticoagulant to 0.1% by mass, and the PEG surface was modified. A ribosome encapsulated hemoglobin preparation was obtained. Hemoglobin concentration in the hemoglobin preparation of each of the thus obtained fatty acid-containing ribosome-produced hemoglobin preparations was measured by atomic absorption, and HSPC concentration, cholesterol concentration and stearic acid concentration were measured by high performance liquid chromatography.

From this, the yield of the protein and the amount of the pulsed protein per lipid amount were determined in the same manner as in Example 1 (FIG. 4).

The results show that changing the number of carbon atoms to 12-18 slightly improves the protein yield, but the amount of encapsulated protein per lipid amount tends to decrease. There is not much effect by changing the number of carbon atoms as compared with the case where the content molar ratio is 30 mol% or more, and when the molar ratio of fatty acid to phospholipid is in the range of 30 mol% to 50 niol%, the effect of the difference in carbon chain length is not affected. It turned out to be small. In the examples, the yield, the ratio per lipid amount, and the leakage rate of the protein as the substance incorporated into the ribosome are measured. However, the same applies when a substance other than the protein is incorporated. is there. Industrial applicability

As described above in detail, the ribosome preparation of the present invention provides a drug-retaining ribosome preparation having an excellent drug retention rate inside the ribosome. Furthermore, they found that ribosomes could be formed by combining fatty acids in amounts previously considered impossible to form ribosomes as higher saturated fatty acids of a specific chain length. Phospholipids are the most frequently incorporated substances in the composition of liposome-constituting membrane materials, and are very expensive compared to fatty acids. According to the present invention, the amount of the inexpensive fatty acid can be increased, and the amount of the phospholipid can be much reduced. Therefore, it is inexpensive as a raw material for ribosome preparations. The present invention provides a film agent having a suitable composition.

Claims

The scope of the claims

1. A ribosome preparation comprising a ribosome and a substance incorporated into the ribosome, wherein the membrane agent constituting the ribosome contains a phospholipid and at least one kind of a higher saturated fatty acid having 10 to 20 carbon atoms; Liposome preparation, characterized in that the content of the liposome is 30 mol% to 50 mol% in the liposome constituents in a molar ratio.

2. The ribosome preparation according to claim 1, wherein the phospholipid is a saturated phospholipid or a hydrogenated phospholipid.

3. The liposome preparation according to claim 1, further comprising cholesterol as the membrane agent.

4. An agglutination inhibitor having a hydrophilic polymer chain portion and a hydrophobic portion is further bound to the ribosome surface. The agglutination inhibitor has a hydrophilic portion immobilized on the lipid layer in the ribosome and becomes hydrophilic. The ribosome preparation according to any one of claims 1 to 3, wherein the hydrophilic polymer chain portion extends outward from the liposome surface.

5. The liposome preparation according to any one of claims 1 to 4, wherein the substance incorporated into the liposome is a physiologically active substance.

6. The liposome preparation according to claim 5, wherein the physiologically active substance is hemoglobin.