JPH0512352B2 - - Google Patents

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a novel liposome,
More specifically, when liposomes are used as pharmaceutical compositions such as pharmaceuticals and cosmetics, liposomes are stable against a wide range of PH changes, liposomes can adjust the external fluidity of the membrane, and fusion between liposomes is possible. The present invention provides a novel liposome capable of

(Prior art) Recently, efforts have been made to make drugs that are difficult to dissolve in water in vivo absorbable, to control the in vivo metabolism, absorption, and excretion of drugs, to improve drug stability, and to slow down drug release. Liposomes using phospholipids and the like are attracting attention for the purpose of release and persistence.

(Problems to be Solved by the Invention) However, liposomes themselves have poor stability, and when liposome membrane forming materials such as dipalmitoylphosphatidylcholine are used, they are expensive; Another problem is that there are concerns about the supply amount if it is industrialized.

(Means for Solving the Problems) As a result of extensive research in order to solve the above problems, the present inventors have discovered purified egg yolk phospholipids and/or
Alternatively, if phosphatidylcholine separated from egg yolk phospholipid is hydrogenated and the degree of hydrogenation is appropriately selected, or if liposomes are produced from a mixture of these, liposomes that do not have various problems can be produced. The present inventors have discovered that the following can be obtained, and have completed the present invention.

The present invention will be explained in detail below.

Natural egg yolk phospholipids contain unsaturated fatty acid residues in their molecules, but in the present invention, hydrogenated phospholipids in which the unsaturated fatty acid residues of the natural egg yolk phospholipids are partially or completely saturated with hydrogen are used. is used as a liposome film-forming material.

The purified egg yolk phospholipid used in the present invention is a purified phospholipid extracted from egg yolk, and has a phospholipid purity of 93% or more. The phospholipid is a mixture of various phospholipids as shown below, and also contains about 2% cholesterol. The phospholipids contained are 64-83% phosphatidylcholine, 22-14% phosphatidylethanolamine, and a small amount of lysophosphatidylcholine, depending on the analysis method.
There is very little sphingomyelin.

In addition, the egg yolk phosphatidylcholine used in the present invention can be obtained from the above-mentioned egg yolk phospholipids by column chromatography or column chromatography using activated alumina or silica gel, etc., using an organic solvent that dissolves the egg yolk phospholipids, such as chloroform-methanol. It is obtained by stirring and mixing egg yolk phospholipids and activated alumina or silica gel, etc., and separating them using the batch method. Although the purity depends on the analysis method, it is over 97% phosphatidylcholine, and contains small amounts of sphingomyelin, lysophosphatidylcholine, and neutral lipid triglycerides. and,
This substance has different types of fatty acid residues (number of carbon atoms,
It is a mixture containing (number of unsaturated bonds, position of unsaturated bonds) in the molecule.

The hydrogenated phospholipid used in the present invention is the purified egg yolk phospholipid or the egg yolk phosphatidylcholine disclosed in Japanese Patent Application Laid-open No. 58-201948 and Japanese Patent Application Laid-open No. 60-1989.
It was hydrogenated according to the method disclosed in Publication No. 105686. The degree of hydrogenation can be adjusted by hydrogen pressure, catalyst amount, reaction time, and reaction temperature, and is determined by the iodine value.

The film-forming material of the present invention contains sterols such as cholesterol as a membrane stabilizer, dicetyl phosphate, phosphatidic acid, etc. as a charged substance, and α- as an antioxidant, according to a conventional method. Tocopherols and the like may also be added.

There are no particular restrictions on the drugs that can be contained in the liposomes of the present invention, as long as they do not inhibit the formation of liposomes. Drugs that are commonly used in pharmaceuticals, quasi-drugs, and cosmetics, such as proteins, water-soluble vitamins, and fat-soluble vitamins, can be used regardless of whether they are water-soluble or fat-soluble.

The liposome of the present invention is produced by a method known per se. For example, a hydrogenated purified egg yolk phospholipid and, if desired, a sterol and a substance imparting a charge are dissolved in an appropriate solvent such as chloroform or ethanol, and the solvent is distilled off from the resulting solution to prepare a phospholipid film. The obtained membrane is dissolved in a suitable solvent such as chloroform or ether, and an aqueous solution of the solvent (if the drug is water-soluble) is added to this solution.
The resulting mixture is vigorously shaken, preferably sonicated, to uniformly disperse the aqueous drug solution. The solvent is distilled off from the dispersion to obtain a drug-retaining liposome.

If the drug is water-insoluble, dissolve the liposome film-forming material (phospholipid) and the drug in an organic solvent,
Next, after the solvent is distilled off to form a lipid thin film, water or an aqueous solution (including an aqueous buffer solution) is added, shaken and stirred, and further subjected to ultrasonication to obtain a drug-retaining liposome.

(Effects of the Invention) The liposome of the present invention can be stable over a wide pH range. In addition, it is possible to obtain membrane fluidity that increases at a specific pH in the weakly acidic region (tumors, inflammation, etc. whose pH is slightly lower than that of normal tissue).
It may be used as a targeting agent to effectively release the drug at the site of infection). moreover,
A stable polymer can be obtained that can significantly change the fluidity of liposome membranes. Then, it is possible to obtain a liposome that promotes fusion between liposomes.

(Examples) The present invention will be further explained by Examples and Test Examples.

Examples 1 to 3 and Comparative Example 1 Hydrogenated purified egg yolk phospholipids with different iodine values (iodine value = 27, 20, 1) and natural purified egg yolk phospholipids (iodine value = 78) as a comparative example were each dissolved in chloroform. (17mg/ml) and this solution
25 ml and 3 μ methanol solution of 5-doxyl stearic acid (1 x 10 ^-3 ml/), which is a spin labeling agent.
were mixed, sonicated for 2 minutes, and cooled on an ice bath for 30 minutes. Furthermore, evaporation was performed without heating under nitrogen gas to distill off the solvent. Thereafter, 20 ml of diethyl ether and 10 ml of distilled water are added, followed by ultrasonication for 1 minute, followed by evaporation under nitrogen gas to form a thin layer of purified egg yolk phospholipid.
Then, 25 ml of distilled water was added and sonicated for 10 minutes to obtain unilamellar giant liposomes.

Test Example 1 The pH of the liposome solutions obtained in Examples 1 to 3 and Comparative Example 1 was adjusted with HCl or NaOH (2
<PH<12), and when the fluidity of the membrane was measured using an electron spin resonance apparatus, the relationship between the order parameter (S) and PH as shown in Figure 1 was obtained.

(Here, the “S value” is a parameter that indicates the fluidity of the phospholipids that make up the membrane, and is generally referred to as an order parameter. This value is a value in the range of 0≦S≦1. When S = 0, it means that the spin label agent is rotating freely and extremely quickly, and when S = 1, it means that its movement is completely fixed like a crystal. (In other words, the larger the S value, the more restricted the movement of phospholipids in the membrane is, meaning that the fluidity of the entire membrane is lower and more stable.) The smaller the value, the larger the S value, indicating that the fluidity of the membrane is lower. Furthermore, while saturated phospholipids have almost no pH dependence, as the degree of unsaturation increases, the S value decreases on the low pH side and the fluidity of the membrane increases.

Examples 4, 5 and Comparative Example 2 Hydrogenated egg yolk phosphatidylcholine (iodine value =
20,2) and egg yolk phosphatidylcholine (iodine value = 67) extracted from egg yolk as Comparative Example 2.
Liposome solutions were prepared in the same manner as in Examples 1 to 3 and Comparative Example 1 above.

Test Example 2 When the membrane fluidity of the samples obtained in Examples 4 and 5 and Comparative Example 2 was measured using electron spin resonance in the same manner as in Test Example 1, the S value and PH were as shown in Figure 2. The following relationship was obtained.

Regardless of the composition of the phospholipid, the same results as Test Example 1 were obtained.

Test Example 3 A liposome solution consisting of hydrogenated purified egg yolk phospholipid with an iodine value of 20 from Example 2 (PH = 6.0, S =
0.63), polycations, which are generally known as polymers that strongly interact with such liposomes and can greatly change the fluidity of the membrane (polymer having cation sites in the chain, present example) So poly[(dimethyliminio)ethylene(dimethyliminio)methylene-paraphenylene dichloride]
When added within the range of 0.005 to 10% by weight,
The S value changed only by ±0.02. The fluidity of the membrane did not change much, indicating that it was stable.

Test Example 4 The effect of water-soluble polymer polyethylene glycol diamine 10 (PGD10) on homofusion of liposomes in Examples 2 and 3 and Comparative Example 1 was investigated.
The results are shown in Figure 3. Liposome fusion was evaluated by quenching rate EQ using a fluorescence quenching method using liposomes labeled with fluorescamine (fluorescent agent) and trinitrobenzenesulfonic acid (quencher).

The maximum quenching rate increases as the number of double bonds in the phospholipid decreases. In other words, it was found that saturation of the double bonds in phospholipids promoted fusion.

[Brief explanation of the drawing]

Figures 1 and 2 are graphs showing the relationship between the membrane fluidity and PH of the liposomes of the present invention, and Figure 3 is the fusion rate and polyethylene glycol diamine (PGD) 10
It is a graph showing the relationship between the concentration of

Claims (1)

[Claims] 1. A liposome whose membrane-forming material is hydrogenated purified egg yolk phospholipid, and whose PH stability is increased depending on the degree of hydrogenation. 2. The liposome according to claim 1, wherein the hydrogenated purified egg yolk phospholipid is hydrogenated egg yolk phosphatidylcholine. 3. The liposome according to claim 1, which has a high degree of hydrogenation and is stable over a wide pH range. 4. The liposome according to claim 1, which has a partial degree of hydrogenation and has increased membrane fluidity in an acidic region.