JPS6354684B2 - - Google Patents

Description

Detailed Description of the Invention Background of the Invention (Technical Field) The present invention provides improved drug-retaining liposome preparations, more specifically drug-retaining liposomes in which various physiologically active substances are encapsulated inside the liposomes (endoplasmic reticulum). Concerning improved formulations. Liposomes are defined as closed vesicles with membranes consisting primarily of lipids. Drug-retaining liposome preparations may be used to stabilize unstable drugs and to provide sustained drug release in vivo, and may also be used as a means to selectively transfer drugs to specific organs. It is being For example, liposome formulations containing insulin, heparin, urokinase, etc. are known as formulations for stabilization and sustained release, and formulations for rapid drug transfer to target organs include iupidecarenone, cytosine, etc. Liposome preparations containing arapinoside and the like are known. (Prior art and problems) Phospholipids used as membrane materials for conventional drug-retaining liposome preparations include phosphatidylcholine,
Products derived from egg yolk, soybeans and other animal tissues such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, egg yolk lecithin or soybean lecithin which is a mixture of these, dipalmitoyl lecithin, distearoyl Examples include synthetic lecithins such as lecithin. These have good affinity for living organisms and can be safely used in oral or parenteral preparations such as injections. However, conventionally used liposome membrane materials do not necessarily have a sufficient drug retention rate. That is, the amount of drug that can be encapsulated in a unit liposome membrane material is not sufficient, and a more efficient membrane material is desired. In addition, conventional liposome membrane materials have insufficient stability and have the disadvantage that the membrane is relatively easily destroyed in vitro or in vivo, releasing the drug.
Furthermore, synthetic lecithin is very expensive and difficult to obtain, making it unsuitable as a pharmaceutical raw material. Purpose of the Invention Therefore, the first purpose of the present invention is to provide a drug-retaining liposome preparation that has good affinity for living organisms and can be safely administered orally, transdermally, subcutaneously, intravenously, rectally, etc. be. A second object of the present invention is to provide a drug-retaining liposome preparation that has an excellent drug retention rate inside the liposome. A third object of the present invention is to provide a method in which the liposome membrane is in
The object of the present invention is to provide a drug-retaining liposome preparation that is stable in vitro and in vivo and has excellent sustained drug release properties. Detailed Description of the Invention As the membrane material used in the drug-retaining liposome preparation of the present invention, a suitable lipid is used to prepare the liposome. In particular, phospholipids include natural phospholipids such as soybean lecithin and egg yolk lecithin, and synthetic phospholipids. Any phospholipid can be used, such as a lipid or a hydrogenated natural phospholipid. Since all natural phospholipids contain unsaturated fatty acids, in order to achieve the object of the present invention to a higher degree, it is necessary to use hydrogenated phospholipids in which the unsaturated fatty acids of the natural phospholipids are saturated with hydrogen. is more effective. Synthetic phospholipids can also be used, but are currently very expensive and unsuitable for economic reasons. Typical examples of phospholipids used in the present invention include lecithin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, cardiolipin, and the like. Further examples include those obtained by hydrogenating these in accordance with conventional methods. In particular, hydrogenated natural lecithin obtained by hydrogenating soybean lecithin, egg yolk lecithin, corn lecithin, cottonseed oil lecithin, rapeseed lecithin, etc. is preferably used. The higher fatty acids in the present invention include carbon atoms
Higher fatty acids having 10 to 20 fatty acids are preferred, examples of which include capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid,
Examples include linoleic acid, linolenic acid, arachidonic acid, and the like. Particularly preferred are unsaturated higher fatty acids having 1 to 4 double bonds and having 18 to 20 carbon atoms, such as oleic acid, linoleic acid, linolenic acid, arachidonic acid, and most preferred is oleic acid. These higher fatty acids can be used alone or in combination, and the blending amount is preferably 5 wt% or more and a concentration (wt%) at which the phospholipids do not form micelles. When the amount of higher fatty acids blended is 5 wt% or more, more preferably 10 wt% or more, excellent effects are exhibited in terms of affinity, stability, sustained release, etc., which are the objectives of the present invention. Unless phospholipids are used in a concentration range that forms a lamellar structure, drugs cannot be retained in liposomes. When the blending amount of higher fatty acids increases, phospholipids mainly contain higher fatty acids to form micelles, and liposomes are no longer formed. The amount of higher fatty acids blended at this time varies depending on the conditions, but is approximately 30 wt%. Therefore, unless the higher fatty acid is used in an amount less than this amount, liposomes will not be formed, and the drug retention efficiency will be extremely reduced or become impossible. Preferably it is 15 wt% or less. Sterols such as cholesterol and tocopherol can be added to the membrane material of the present invention in order to increase the strength of the membrane, and substances that impart a negative charge to adjust the sustained release of the drug in the living body. For example, phosphatidic acid and dicetyl phosphate can be added. The disintegration of liposome membranes is controlled by the presence of these substances. There are no particular restrictions on the substances to be incorporated and retained in the liposome formulation of the present invention as long as they do not inhibit the formation of liposomes; , or those that are desired to be quickly distributed to specific organs are preferably used. Examples of such retained substances include insulin, heparin, urokinase, iupidecarenone, methotrexate, neomycin,
Examples include bleomycin, tetracycline, cytochrome C, asparaginase, cytosine arabinoside, and the like. In addition, other than drugs,
anchor, plasmid, DNA, RNA, etc.
There is no particular restriction as long as it is effective when administered in vivo. The drug-retaining liposome formulation 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 fatty acids and optionally sterols,
A substance giving a negative charge is dissolved in a suitable solvent such as chloroform or ethanol, and the solvent is distilled off from the resulting solution to prepare a phospholipid film. The resulting membrane is dissolved in an appropriate solvent such as chloroform or ether, an aqueous drug solution is added to this solution, and the resulting mixture is vigorously shaken, preferably ultrasonicated, to uniformly disperse the aqueous drug solution. let The solvent is distilled off from the dispersion to obtain a drug-loaded liposome preparation.
The liposomes thus obtained are washed with a physiologically acceptable aqueous solution, such as physiological saline, if necessary, and then prepared into pellets, suspensions, tablets, capsules, granules, and powders. The liposome preparation of the present invention is administered orally or as a parenteral preparation such as an injection. Furthermore, the liposomes of the present invention may be lyophilized under normal conditions;
For example, it is preferable to freeze at -20 to -80°C and sublimate the ice under reduced pressure of 0.3 torr or less. In order to form a better freeze-dried cake, commonly used excipients such as mannitol, dextrin, glycine, etc. may be added. In the present invention, a liposome preparation is defined as one in which various substances to be retained are sealed inside the liposome. Next, the present invention will be described in more detail with reference to Examples and Test Examples. Example 1 32 mg of lecithin, 15.4 mg of cholesterol and 5 mg of oleic acid were dissolved in 3 ml of chloroform, and 50 ml of
It was placed in an eggplant-shaped flask. 3 ml of isopropyl ether was added to this, and after stirring and mixing, 1 ml of an aqueous solution containing 10,000 units of urokinase was added. This mixed solution was sonicated in a water bath type ultrasonic cleaner at a temperature of 4° C. for 5 minutes to uniformly disperse the mixture. Next, using a rotary evaporator,
The solvent was evaporated on a 40°C water bath until a gel was formed. After adding 5 ml of physiological saline to the formed gel and stirring, solvent distillation was continued for another 10 minutes to obtain a liposome suspension. This suspension was centrifuged at 100,000G for 30 minutes and washed twice with physiological saline. The obtained pellets were suspended in physiological saline and sterilized to obtain urokinase-retaining liposomes. For comparison, lecithin 32mg, cholesterol
15.4 mg and 3.1 mg of dicetyl phosphate were dissolved in 15 ml of chloroform, placed in a 50 ml eggplant-shaped flask, and the solvent was distilled off using a rotary evaporator to form a thin film on the inner wall of the flask. Next, the mixture was dissolved in 3 ml of chloroform, 3 ml of isopropyl ether was added, and the mixture was stirred. The procedure was then carried out in the same manner as above to obtain a urokinase-retaining liposome for comparison. Example 2 Instead of the urokinase aqueous solution of Example 1, 1 ml of an aqueous solution containing 1000 units of heparin was added, and Example 1
Heparin-retaining liposomes of the present invention and for comparison were prepared in the same manner as above. Example 3 Instead of the urokinase aqueous solution of Example 1, 1 ml of an aqueous solution containing 40 units of insulin was added, and Example 1
Insulin-retaining liposomes of the present invention and comparison were prepared in the same manner as above. Example 4 Instead of the urokinase aqueous solution of Example 1,
1 ml of 0.3M glucose aqueous solution was added and prepared in the same manner as in Example 1 to obtain glucose-retaining liposomes for the present invention and for comparison. Example 5 Oleic acid was used instead of oleic acid in Example 1.
Example 1
Urokinase-retaining liposomes for the present invention and for comparison were prepared in the same manner as above. The amount of drug entrapped within the liposomes of the thus obtained liposome formulation was measured. The results are shown in Table 1. The drug retention rate was calculated using the following formula. Drug retention rate (%) = Amount of drug captured in liposomes / Amount of drug used during preparation × 100 As is clear from Table 1, by blending higher fatty acids with phospholipids as in the present invention, It can be seen that the drug retention rate is significantly improved in both egg yolk lecithin and hydrogenated egg yolk lecithin.

【table】

[Table] Next, in the urokinase-retaining liposome
Vitro and in vivo stability tests were performed. Experimental Example 1 In vitro stability test of urokinase-retaining liposomes Urokinase-retaining liposomes prepared according to Example 1 were tested according to the 10th revised Japanese Pharmacopoeia disintegration test method, liquid 1 (artificial gastric fluid), liquid 2 (artificial intestinal fluid). and suspended in 90% rabbit serum and incubated at 37°C.
It was investigated over time using the 2444 method. The results are shown in FIGS. 1, 2 and 3, respectively. In Fig. 1, Fig. 2, and Fig. 3, the lines marked with ● use hydrogenated egg yolk lecithin as the phospholipid;
The line marked ■ indicates the case of the liposome of the present invention using egg yolk lecithin, the line marked ○ shows the case of the liposome of the comparative example using hydrogenated egg yolk lecithin, and the line marked □ shows the case of the liposome of the comparative example using egg yolk lecithin. FIG. 1 shows the stability in the first liquid, and it was confirmed that the liposome of the present invention has increased stability compared to the comparative liposome. FIG. 2 shows the stability in the second liquid, and it was confirmed that the liposome of the present invention has increased stability compared to the comparative example liposome. FIG. 3 shows the stability in rabbit serum, and it was confirmed that the liposome of the present invention has increased stability compared to the comparative liposome. Experimental Example 2 In vivo stability test of urokinase-retaining liposomes 1000 units of each of the urokinase-retaining liposomes prepared according to Example 1 were injected into male Wistar rats weighing 200 to 250 g through the tail vein, and 30 and 60 days after administration. , 120, and 180 minutes later, blood was collected, and urokinase activity in the blood was examined using the synthetic substrate S-2444 method. The results are shown in FIG. In FIG. 4, the ● marked line, ■ marked line, ○ marked line, and □ marked line have the same meanings as shown in FIGS. 1 to 3. As is clear from FIG. 4, the liposome preparation of the present invention has extremely good stability in blood and exhibits unprecedented effectiveness. Effects of the Invention The drug-retaining liposome formulation according to the present invention provides many advantages over conventional formulations as described below. (1) First of all, the liposome preparation of the present invention has good affinity for living organisms and can be safely administered orally, transdermally, subcutaneously, intravenously, rectally, etc. (2) A drug-retaining liposome preparation with an excellent drug capture rate inside the liposome is provided. (3) The liposome membrane of the present invention can be used in vitro and in vitro.
It is stable in vivo and has excellent sustained drug release properties.

[Brief explanation of drawings]

Figures 1, 2, and 3 show the results of the disintegration test method of the 10th revised Japanese Pharmacopoeia, liquids 1 and 2, and 90% domestic rabbit serum for the liposome formulation of the present invention and the liposome formulation of the comparative example, respectively. Graph comparing stability,
FIG. 4 is a graph comparing the stability in vivo when administered intravenously to rats.

Claims (1)

[Scope of Claims] 1. In a liposome preparation consisting of a liposome and a substance incorporated into the liposome, the membrane material constituting the liposome contains a lipid and at least one
1. A liposome preparation containing 5 to 15 wt% of an unsaturated higher fatty acid having 18 to 20 carbon atoms and having 1 to 4 double bonds. 2. The liposome preparation according to claim 1, wherein the lipid is a hydrogenated phospholipid.