JPS60155109A - Liposome pharmaceutical - Google Patents

Abstract

PURPOSE:The titled stable pharmaceutical, obtained by incorporating a higher fatty acid with a lipid used as a membrane material, and having improved affinity for the living body, retention of chemicals in the interior thereof and sustained release property of the chemicals. CONSTITUTION:A liposome pharmaceutical obtained by incorporating >=5wt%, preferably 10-15wt% higher fatty acid with a lipid, particularly phospholipid as a membrane material in a liposome pharmaceutical for retaining chemicals. A 10-20C higher fatty acid is preferred for the higher fatty acid, and particularly an 18-20C unsaturated higher fatty acid (optimally oleic acid) having 1-4 double bonds is preferred. A hydrogenated natural lecithin such as hydrogenated soybean lecithin, egg yolk lecithin or corn lecithin is preferably used as the phospholipid.

Description

Detailed Description of the Invention 11. Background of the Invention (Technical Field) The present invention provides an improved drug-retaining liposome formulation, more specifically, a drug-retaining liposome in which various physiologically active substances are encapsulated inside the liposome (endoplasmic reticulum). The present invention relates to an improved formulation of.

Liposomes are defined as closed vesicles with membranes consisting primarily of lipids. Drug-retaining liposome preparations can be used to stabilize unstable drugs and slow release of drugs in vivo, and can also be used as a means to selectively transfer drugs to specific organs. For example, preparations aimed at stabilization and sustained release include insulin, heparin, and chloride. Liposome preparations containing kinases, etc. are known, and are used as preparations for the purpose of rapid transfer of drugs to target organs. Liposomal preparations containing ubidecarenone, cytosine arapinoside, □do, etc. are known.

(Prior art and issues).

Phospholipids used as membrane materials for conventional drug-retaining liposome preparations include those derived from egg yolk, soybeans, and other animal tissues, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin, and mixtures thereof. Examples include egg yolk lecithin, soybean lecithin, and synthetic lecithins such as dipalmitoyl lecithin and distearoyl 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 are
Drug retention rate is not always sufficient. 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;
It has the disadvantage that the membrane is relatively easily destroyed in vitro or in vitro, releasing the drug.

Furthermore, synthetic lecithin is very expensive and difficult to obtain.
Not suitable as a pharmaceutical raw material.

II. OBJECTS OF THE INVENTION Accordingly, the first object 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. It is in.

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 liposome membrane in vitro.
Another object of the present invention is to provide a drug-retaining liposome preparation that is stable in vitro and has excellent sustained drug release properties.

■1 Detailed description of the invention As the membrane material used in the drug-retaining liposome preparation of the present invention, lipids suitable for making liposomes are used, and in particular, phospholipids include natural phospholipids such as soybean lecithin and egg yolk lecithin. Any phospholipid can be used, such as synthetic phospholipids or hydrogenated natural phospholipids. 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, calciolipin, 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 acid in the present invention has 10 to 2 carbon atoms.
Higher fatty acids having 0 fatty acids are desirable, examples of which include capric acid, lauric acid, myristic acid, palmitic acid,
Examples include stearic acid, oleic acid, linoleic acid, lylunic 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, linolic acid, and arachidonic acid, and oleic acid is most preferred.

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 blending amount of higher fatty acids 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. As the amount of higher fatty acids increases, phospholipids become higher
It mainly contains fatty acids and forms micelles and no longer forms liposomes. 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 impossible. Preferably 15wt
% 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 in order to reduce the sustained release of the drug in the living body by 1, negative electric current can be added. It is possible to add substances such as phosphatidic acid, dicetyl phosphate. 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 preparation of the present invention as long as they do not inhibit the formation of liposomes, but
Preferably used are those that are unstable in terms of immobility, are gradually released within the body, or are desired to be quickly distributed to specific organs. Examples of such retained substances include insulin, heparin, urokinase, ubidecarenone, methotrexate, and neomycin.

Bleomycin, tetracycline, cytochrome C,
Examples include asparaginase and cytosine arabinoside. Anything other than drugs.

There are no particular restrictions on the marker, plasmid, DNA, RNA, etc. as long as it is effective when administered in vivo.

The drug-retaining liposome 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 fatty acids, and optionally sterols, a substance imparting a negative charge, are mixed in chloroform. , dissolved in an appropriate solvent such as ethanol, and distilling off the solvent from the resulting solution to prepare a phospholipid film. The obtained membrane is dissolved in an appropriate solvent such as chloroform or ether, an aqueous drug solution is added to this solution, the resulting combined solution is shaken vigorously, and preferably subjected to ultrasonication to dissolve the aqueous drug solution. The solvent is distilled off from the uniformly dispersed dispersion to obtain a drug-retaining liposome preparation.

The liposomes thus obtained are physiologically acceptable if necessary6. After washing with an aqueous solution (e.g. 12 physiological saline), the liposome preparation of the present invention can be prepared into pellets, suspensions, tablets, capsules, granules, powders, etc. Administered as an oral formulation. Also,
The liposomes of the present invention may be freeze-dried under normal conditions, such as freezing at -20 to -80°C, Q
-3 torr or less. Sublimation of reduced JETE ice, = 6° good.

In order to form a better freeze-dried product, commonly used excipients such as mannitol, dextrin, glycine, etc. may be added.

In the present invention, a liposome preparation is defined as a liposome preparation in which various substances to be retained are encapsulated inside the liposome.

Next, the present invention will be explained in further detail by showing Examples and Test Examples.

[Example 1] 321 g of lecithin, 15.4 mg of cholesterol, and 5 g of oleic acid were dissolved in chloroform,
The mixture was placed in a 50 liter eggplant-shaped flask. 3 ml of isopubyl ether was added to this, and after stirring and mixing, 1 portion of water infusion containing 10,000 units of urokinase was added. Then, using a rotary evaporator, dissolve the gel on a 40°C water bath until it forms a gel, and add physiological saline to the formed gel. After adding 5-1 of water and stirring, solvent distillation was continued for another 10 minutes to obtain a liposome suspension.
- Centrifuged at 00,000 G for 30 minutes and washed twice with physiological saline. The resulting beret was soaked in physiological saline for 5W!
A urokinase-retaining ribbon was obtained by making it cloudy and sterilizing it. □ For comparison □, lecithin 32. , -g, cholesterol-
□Le 15-4. mg and Giseti J Rephosphate 3
．． 1mz was dissolved in chloroform 151, put into a 501 volume eggplant flask, and the solvent was distilled off using a 40-meter tally evaporator to form a thin film on the inner wall of the flask. Then, add □isopropyle, -chill 31 and stir to mix, as follows...,'
1,' Same as above - On the other hand, by operating the : method, a comparative urokinase-retaining protein, Ri1, Shi≠mu□ was obtained. [Example 2] Instead of the urokinase aqueous solution of Example 1, heparin 10
1 ml of an aqueous solution containing 0.00 units was added and prepared in the same manner as in Example 1 to obtain heparin-retaining liposomes for the present invention and for comparison.

[Example 3] Instead of the urokinase aqueous solution of Example 1, insulin 4
An aqueous solution 141 containing 0 units was added and prepared in the same manner as in Example 1 to obtain insulin-retaining liposomes for the present invention and for comparison.

[Example 4] Instead of the urokinase aqueous solution in Example 1, 1 ml of a 0.3M glucose aqueous solution was added and prepared in the same manner as in Example 1 to obtain glucose-retaining liposomes of the present invention and for comparison.

[Example 5] Instead of oleic acid in Example 1, 2.5 m of oleic acid
g and 2.5 mg of myristic acid were added thereto and prepared in the same manner as in Example 1 to obtain urokinase-retaining liposomes for the present invention and for comparison.

The amount of drug entrapped within the liposomes of the thus obtained liposome formulation was measured. The results are shown in Table 1.

In addition, the drug retention rate was determined by the following formula.

100 As is clear from Table 1, by blending higher fatty acids with phospholipids as in the present invention, the drug retention rate is significantly improved for both unhydrogenated natural egg yolk lecithin and hydrogenated egg yolk lecithin. I can see that it is being done.

Table 1 Next, in vitro analysis of urokinase-retaining liposomes.
and 1nvi MAO stability tests were performed.

[Experimental Example 1] 1n polymer 1t of urokinase-retaining liposome
Urokinase-retaining liposomes prepared according to ro stability test Example 1 were tested according to the 10th revised Japanese Pharmacopoeia disintegration test method in the first fluid (artificial gastric juice), the second fluid (artificial intestinal fluid), and 90% domestic rabbit serum. The suspension was incubated at 37° C., and the drug residual rate in the liposomes was examined over time using the synthetic substrate S-2444 method. The results are shown in FIGS. 1, 2 and 3, respectively.

In Figures 1, 2, and 3, the lines marked with * are for the case of the liposome of the present invention using hydrogenated egg yolk lecithin as the phospholipid, and the lines marked with ■ are for the case of the liposome of the present invention using egg yolk lecithin as the phospholipid.
The marked line shows the case of a comparative liposome using hydrogenated egg yolk lecithin i, and the stamped line shows the case of a comparative liposome 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 self-destruction, and it was confirmed that the liposome of the present invention has increased stability compared to the comparative example liposome.

[Experimental Example 2] Urokinase-retaining liposome in my IV
o Stability Test 1000 units of each of the urokinase-retaining liposomes prepared according to Example 1 were injected into the tail vein of male Wistar rats weighing 200 to 250 g.
Blood was collected after 60, 120, and 180 minutes, 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 * mark line, the I mark line, the 0 mark line, and the mid-mouth 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.

(1) Effects of the Invention The drug-retaining liposome preparation according to the present invention has many advantages over conventional ones, 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 is stable in v+tro and in imma, and has excellent sustained drug release properties.

[Brief explanation of the drawing]

Figures 1, 2, and 3 show the liposome formulation of the present invention and the liposome formulation of the comparative example in liquids 1 and 2 of the 10th revised Japanese Pharmacopoeia disintegration test and in 90% domestic rabbit serum. Figure 4 is a graph comparing the stability in vivo when administered intravenously to rats. Figure 1 Time (h'') Figure 2 Time (h'') Figure 3 Time (hr) ＼ Figure 4 P. 0 1 2 3 B Temple (hr)

Claims (1)

[Claims] (1) A liposome preparation consisting of a liposome and a substance incorporated into the liposome, characterized in that the membrane material constituting the liposome contains a lipid and at least one type of higher fatty acid. . (2) The liposome preparation according to claim 1, in which the higher fatty acid content is 5 wt% or more... (3) Claim 1, in which the higher fatty acid has 10 to 20 carbon atoms. Or the liposome preparation according to item 2. (4) The liposome preparation according to claim 1, wherein the lipid is a hydrogenated phospholipid.