JPH0457375B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an industrial method for producing liposomes and raw materials thereof.

Liposomes, which are closed vesicles of lipids, have originally been widely used as a biological membrane model, but recently they have been used in various applications for drug delivery. The types of liposomes can be roughly divided into multilamellar liposomes (MLVs), large unilamellar liposomes (LUVs), and small unilamellar liposomes (SUVs), and various preparation methods for each have already been reported (annual, Review of Biophysics and Bioengineering, Volume 9, Page 467 (1980)]. However, these are only preparation methods at the laboratory level on a test tube or eggplant-shaped Kolben scale. This is not a production method that will immediately lead to industrial production.As an industrial production method for liposomes, a method has recently been reported in Japanese Patent Application Laid-Open No. 171915/1982, but this method uses two types of organic solvents in which membrane component substances are dissolved, In other words, it is a method in which a hydrophobic substance and an aqueous solvent-soluble substance are injected into an aqueous solution, which requires the use of a specific combination of organic solvents, and tends to produce monolamellar liposomes mainly made of phospholipids. It is difficult to say that industrial production of liposomes has become possible due to the disadvantages that liposomes with non-uniform particle sizes are likely to be formed and drugs are difficult to be efficiently retained within the liposomes.Therefore, many researchers have It is no exaggeration to say that one of the major reasons why liposome preparations have not yet been commercialized is the difficulty of industrial production, despite clinical application research being carried out.

In view of these circumstances, the inventors conducted intensive studies on industrial methods for producing liposomes, and as a result, they discovered the most widely known vortexing method [Journal of Molecular Biology, Vol. 13, p. 238]. (1965)] is not necessarily necessary to form a clean thin film of phospholipids on a glass wall in the production of liposomes; instead, it is simply necessary to prepare a homogeneous mixture of film component substances and add this to an aqueous solvent. The present inventors have discovered that when swollen into hydrated liquid crystals and sufficiently stirred, liposomes that are surprisingly uniform and have good drug retention efficiency can be produced with good reproducibility, leading to the completion of the present invention.

That is, according to the present invention, a homogeneous mixture is produced by swelling the usual membrane component materials constituting liposomes in a small amount of volatile organic solvent, and then removing the organic solvent while stirring or kneading with a stirrer. death,
Add an aqueous solvent to this to determine the phase transition temperature (Te) of the lipid.
By swelling with the above steps and further finely dispersing it with a stirrer, ribosomes can be produced in large quantities.

The membrane component substances used in the present invention include, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine,
In addition to phospholipids represented by sphingomyelin, egg yolk lecithin, soybean lecithin, etc., one or a mixture of two or more of glycolipids, dialkyl type synthetic surfactants, etc. are mainly used. In addition, sterols such as cholesterol and cholestanol are added as membrane stabilizers, and dicetyl phosphate and charged substances are used as membrane stabilizers.
A membrane component substance may be formed by adding phosphatidic acid, ganglioside, stearyl amyl, etc., and further adding α-tocopherol etc. as an antioxidant. Although the ratio of these liposome membrane component substances should not be limited in any way, it is preferable to add about 0 to 2 parts by weight of sterols and about 0.1 parts by weight of charged substances to 1 part by weight of lipid. .

Chloroform, ether, ethanol, etc. are suitable as volatile organic solvents that swell membrane component substances. These organic solvents may be used alone or in combination, but when used in combination, it is desirable that they be mutually miscible. The usage ratio of these organic solvents to the membrane component materials is 1
It is preferably about 0.1 to 1.6 parts by weight.

In addition, as the aqueous solvent for dispersing the membrane component substance, water, physiological saline, buffer solution, aqueous solution of sugars, a mixture thereof, etc. are preferably used, and the usage ratio with the membrane component substance is 1 part by weight of the membrane component substance. against 10
Approximately 1000 parts by weight is appropriate.

The drugs retained in the liposome preparation of the present invention are not particularly limited, but include cytosin arabinoside, anticancer drugs typified by methotrexate, antibiotics typified by penicillin G, insulin,
Proteins such as interferon and glucoamylase, polysaccharides such as dextran, acids such as DNA and RNA, vitamins such as vitamin A, and general drugs such as sodium salicylate are used. These drugs are used after being dissolved in an aqueous solvent, but it is more efficient for membrane-compatible drugs such as chlorophyll, gramicidin S, vitamin A, etc. to be mixed in an organic solvent together with membrane component substances.

Liposomes can be produced industrially based on the present invention by the following procedure.

First, a membrane component material to which a predetermined amount of component material and optionally a membrane-compatible drug has been added is swollen in a small amount of a volatile organic solvent. This is the first point in which the present invention differs from the vortex swing method of Bangham et al. In other words, the vortex swing method produces a clean thin film (lipid film,
Since it is required to form a lipid film on the glass wall surface, it is essential to completely dissolve the film component substances in an organic solvent. Therefore, the amount of organic solvent used is large, and the ratio of organic solvent to membrane component material is 30 to 100 parts by weight per 1 part by weight of membrane component material.
It is used in the weight section. In the present invention, the volatile organic solvent is simply added to produce a homogeneous mixture in which membrane component substances (for example, lipids, cholesterols, and charged substances) are molecularly dispersed. Therefore, the amount of organic solvent used does not need to be as large as in the vortexing method, and only the minimum amount necessary is enough to allow the membrane component substances to freely mix or be solvated at the molecular level, mix with each other, and swell. Not limited. In other words, whether it is in the form of a clear liquid or paste, an opaque paste or fixed form, or a mixture of these, if the membrane component substances are freely mixed or solvated at the molecular level, the appearance does not matter at all. do not. In general, membrane component substances can be swollen in volatile organic solvents relatively easily, but it is more efficient if means such as heating, stirring, or kneading are used.

The thus obtained swollen membrane component material is completely removed into an organic solvent while stirring or kneading with a stirrer to prepare a homogeneous mixture. The term "homogeneous mixture" as used herein refers to a glass-like or paste-like semi-solid material in which membrane component substances, etc. are molecularly dispersed. This is the second major difference from the vortex swing method. That is, in the vortex swing method, it is required to form a clean thin film of the film component substance on the glass wall surface, but this is not necessarily necessary in the present invention.
However, it is sufficient that the membrane component substances are molecularly dispersed with each other, and appearance is not a problem at all.

In order to completely remove the organic solvent, pressure reduction, inert gas such as nitrogen gas, cleansing dry air, etc. may be used. In this case, it goes without saying that heating is more efficient. Furthermore, when pumping inert gas or cleaning dry air, etc., the gas outlet may be placed on the surface of the organic solvent liquid, but bubbling directly immersed in the organic solvent is more efficient and requires stirring at the same time. You can also do it. The volatile organic solvent removed from the reaction vessel is liquid nitrogen,
It can be recovered by using a trap cooled with acetone-dry ice or the like, and since it can be almost completely recovered, there is no problem in terms of safety.

The thus obtained homogeneous mixture of membrane component substances, etc. is collected as is and subjected to treatments such as nitrogen replacement.
It may be stored at a temperature below 0.degree. C., or the next operation, that is, adding an aqueous solvent to swell it to produce a hydrated liquid crystal, may be continued. This operation of adding an aqueous solvent to cause swelling proceeds quickly by heating the lipid to a temperature higher than the phase transition temperature (Tc) of the lipid.

Once sufficiently swollen, the desired liposome preparation can be produced by sufficiently dispersing it using an emulsifying device commonly used for emulsification, such as a homogenizer or propeller mixer. At this time, it goes without saying that heating above Tc is more efficient.
Also preferably during this swelling and dispersion operation and/or
Alternatively, it is desirable to perform bubbling with an inert gas after the dispersion is completed.

Note that this swelling and dispersion operation is more efficient if it is carried out at a temperature higher than the phase transition temperature (Tα) of the membrane component material as a powder crystal, and it is also possible to start the swelling and dispersion operation after kneading with an aqueous solvent. Very efficient.

In addition, to increase the retention rate of drugs in liposomes within the same formulation, dissolve the prescribed amount of the drug to be retained in a small amount of aqueous solvent, add this to a homogeneous mixture of membrane component substances, swell and disperse, and finally The remaining aqueous solvent may be added to dilute the solution.

To produce liposome preparations with even smaller particle sizes, it is better to use an ultrasonic emulsifier, high-pressure emulsifier, etc., or to make the particle size uniform, ultrafiltration membrane methods such as polycarbonate membrane filters can be used. It is also possible to control the diameter distribution.

In this way, liposome preparations with a uniform particle size holding a drug can be obtained in large quantities with good reproducibility.
This liposome preparation may be used as it is, or it may be used after separating and removing the drug that is not retained in the liposomes by means such as dialysis, gel filtration, or centrifugation.

EXAMPLES Next, the present invention will be illustrated by Examples, but these Examples are not intended to limit the present invention in any way.

Reference example 1 Fully hydrogenated purified egg yolk lecithin (IV = 1, phospholipid 99% or more, Tc = 45-60°C, Tmax = 52°C) 28.0
g, cholesterol 15.5 g, and dicetyl phosphate 2.2 g were weighed out and dissolved in 100 ml of chloroform in an Ajihomo mixer, and the solvent was removed by supplying nitrogen gas while stirring with a paddle mixer. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen. The temperature inside the Ajihomo mixer at this time was between 50 and 60°C.

To the thus obtained dried pasty homogeneous mixture was added 0.28M glucose aqueous solution 2, which had been kept at 60°C in advance, to sufficiently swell it. When the temperature was maintained between 50 and 60° C. and thoroughly stirred using a homomixer and a paddle mixer and returned to room temperature, a milky white liposome suspension containing glucose was obtained.

0.5 ml of this liposome suspension was taken and subjected to gel filtration using Sephadex G-50 (1 cmφ x 18 cm, physiological saline) to separate and remove glucose not retained in the liposomes. Next, glucose in the liposome fraction was extracted into the aqueous layer by oil/water partitioning according to a conventional method and quantified, and the retention rate was 31.4%.
It was hot.

In addition, when the liposome fraction obtained by gel filtration was observed using an optical microscope (wide-field microscope),
The particles had a uniform spherical shape with a particle size of 1 to several μm.

Reference example 2 Partially hydrogenated purified egg yolk lecithin (IV = 20, phospholipid 99% or more, Tc = 5-50°C, Tmax = 35°C) 28.0
g, cholesterol 7.7 g, and dicetyl phosphate 2.2 g were weighed out and dissolved in 100 ml of chloroform in an Ajihomo mixer, and the solvent was removed while bubbling with nitrogen gas. Below, in the same manner as in Reference Example 1, instead of the glucose aqueous solution, 0.5%
When carried out using sodium salicylate physiological saline solution 2, a milky white liposome suspension retaining sodium salicylate was obtained.

0.5 ml of this liquid was taken and gel filtrated (5°C) in the same manner as in Reference Example 1 to determine the retention rate, which was 27.1
It was %.

Reference Example 3 In the same manner as Reference Example 1, 1% dextran T40 physiological saline solution 2 was added instead of the glucose aqueous solution.
Prepared using However, the solvent was removed under reduced pressure using a rotary vacuum pump, and stirring after addition of the aqueous solvent was performed using a homodisper and a paddle mixer.

A milky white liposome suspension containing dextran T40 was thus obtained.

1 ml of this liposome suspension was taken and subjected to gel filtration using Sepharose CL-4B (2.2 cmφ x 42 cm, physiological saline) to separate and remove dextran T40 that was not retained in the liposomes. Next, dextran T40 from the liposome fraction was mixed with oil/
When extracted into the aqueous layer by water partition and quantitatively determined, the retention rate was 10.4%.

Reference Example 4 28.0 g of fully hydrogenated purified egg yolk lecithin, 7.4 g of cholesterol, and 1.1 g of stearylamine were weighed out and dissolved in 100 ml of chloroform in an Ajihomo mixer, and the solvent was removed while bubbling with nitrogen. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen. The temperature inside the Ajihomo mixer at this time was between 50 and 60°C.

A 1% dextran T40 physiological saline solution 2, which had been kept at 60°C in advance, was added to the dried paste-like homogeneous mixture thus obtained, and the mixture was sufficiently swollen. When the temperature was maintained between 50 and 60°C and sufficiently stirred using a homodisper and a paddle mixer, and the temperature was returned to room temperature, a milky white liposome suspension containing dextran T40 was obtained.

1 ml of this liquid was taken and subjected to gel filtration in the same manner as in Reference Example 3 to determine the retention rate, which was 11.2%.

Reference Example 5 The same formulation as in Reference Example 4 was used, except that dextran T40 was added as a high concentration physiological saline solution, mixed with the membrane component material, and then physiological saline was added and stirred.
That is, a solution was prepared by dissolving 20 g of dextran T40 in 260 ml of physiological saline, and the solution was heated to 60°C in advance. When this and a homogeneous mixture were sufficiently kneaded with a homodisper at around 60°C, a milky white product was obtained. A paste was obtained. Next, add 1740 ml of physiological saline pre-warmed to 55℃ to this paste.
was added, thoroughly stirred using a homodisper and paddle mixer, and returned to room temperature.
A milky white liposome suspension containing T40 was obtained.

1 ml of this liquid was taken and subjected to gel filtration in the same manner as in Reference Example 3 to determine the retention rate, which was 26.7%.

Reference Example 6 Weigh out 280 g of fully hydrogenated purified egg yolk lecithin, 155 g of cholesterol, and 22 g of dicetyl phosphate,
It was completely dissolved in 1000 ml of chloroform in an Ajihomo mixer. The temperature inside the Ajihomo mixer is
The solvent was removed by nitrogen gas bubbling while keeping the temperature between 50-60°C. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen.

A 0.28M aqueous glucose solution 20, which had been kept at 60°C in advance, was added to the dried paste-like homogeneous mixture thus obtained and allowed to swell sufficiently.
When the temperature was maintained between 50 and 60° C. and thoroughly stirred using a homomixer and a paddle mixer and returned to room temperature, a milky white liposome suspension containing glucose was obtained.

0.5 ml of this liquid was taken and subjected to gel filtration in the same manner as in Reference Example 1 to determine the retention rate, which was 32.5%.

Example 1 The same recipe as in Reference Example 1 was used, but the membrane component material was swollen with a smaller amount of chloroform than in Reference Example 1. That is, after a predetermined amount of membrane component material was sufficiently swollen in 40 ml of chloroform, the solvent was removed by nitrogen gas bubbling. Below, in the same manner as in Reference Example 1, using 0.28M glucose aqueous solution 2,
A milky white liposome suspension retaining glucose was obtained.

0.5 ml of this liquid was taken and subjected to gel filtration in the same manner as in Reference Example 1 to determine the retention rate, which was 14.3%.

Furthermore, when the liposome fraction obtained by gel filtration was observed using a wide-field optical microscope, the average particle size was found to be
Relatively large particles, around 1 μm in diameter, were also observed here and there.
This large one had an onion-like structure.

Example 2 A product was prepared using the same formulation as Reference Example 2 and the same preparation method as Reference Example 1 using 0.5% sodium salicylate physiological saline solution 2.

A milky white liposome suspension containing sodium salicylate was thus obtained.

0.5 ml of this liquid was taken and subjected to gel filtration in the same manner as in Reference Example 2 to determine the retention rate, which was 13.9%.

Claims (1)

[Claims] 1. A liposome membrane component material consisting of a phospholipid and two or more additives selected from a charged substance, a membrane stabilizer, and an antioxidant is swollen in a volatile organic solvent, and then stirred or A method for producing liposomes, which comprises removing an organic solvent while kneading to prepare a homogeneous mixture, and adding an aqueous solution to the mixture for dispersion. 2. After swelling a liposome membrane component material consisting of two or more additives selected from a charged substance, a membrane stabilizer, and an antioxidant and a phospholipid in a volatile organic solvent, the organic solvent is added while stirring or kneading. A homogeneous mixture obtained by removing.