JPS62152531A - Preparation of liposome - Google Patents

Abstract

PURPOSE:To make it possible to industrially prepare a liposome, by spray-drying a solution prepared by dissolving a liposome membrane componential substance in a volatile org. solvent to prepare a uniform mixture and subsequently dispersing said mixture throughout an aqueous solvent. CONSTITUTION:A liposome membrane componential substance pref. consisting of 1pt.wt. of (a) lipid (e.g., phosphatidylcholine), 0-1pt.wt. of (b) sterols (e.g., cholesterol) and 0-0.2pt.wt. of (c) a charge substance (e.g., ganglioside) is dissolved in a volatile org. solvent to prepare a solution which is, in turn, spray- dried to obtain a uniform mixture wherein the liposome membrane componential substances are uniformly mixed. Subsequently, an aqueous solvent is added to said uniform mixture to swell the membrane componential substances and these substances are sufficiently dispersed using an emulsifying apparatus to prepare a liposome suspension. In dissolving the membrane componential substances in the volatile org. solvent, if a nuclear substance (e.g., sorbitol) insoluble in the volatile org. solvent is added, a powder having a particle size still more small is often obtained.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to a novel method for producing ribosomes.

Ribosomes, which are closed lipid vesicles, have been widely used as a biological membrane model to study their physicochemical properties.
On the other hand, since ribosomes can hold various drugs inside them, many studies have been conducted on their use as drug carriers.

<Description of prior art> As a method for producing ribosomes, recently, Japanese Patent Application Laid-Open No. 60-793
No. 2, No. 60-7933, No. 50-7934 and No. 60
Although methods such as No.-12127 have been reported, the number thereof is very small. Although many researchers are conducting clinical application research on ribosomes,
It is no exaggeration to say that one of the major reasons why ribosomal preparations have not yet been commercialized is the difficulty of industrial production.

<Problems to be Solved by the Invention> As a result of intensive studies on the industrial production method of ribosomes, the present inventors found that an amorphous homogeneous mixture can be obtained by spray drying a solution consisting of ribosome membrane component substances and a volatile organic solvent. They also discovered that the homogeneous mixture quickly swells in an aqueous solvent and is further dispersed to produce ribosomes that are surprisingly uniform and have good drug retention efficiency, and have completed the present invention.

<Configuration of the Invention> The present invention is characterized in that a homogeneous mixture prepared by dissolving a ribosome membrane component substance in a volatile organic solvent and then removing the organic solvent by spray drying is dispersed in an aqueous solvent. Open to methods for producing ribosomes.

Ribosome membrane component substances include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, glycolipids, dialkyl type synthetic surfactants, etc. The main component is one or a mixture of two or more of the following. Then, sterols such as cholesterol and cholestanol are added as membrane stabilizers, dicetyl phosphate, phosphatidic acid, ganglioside, stearylamine, etc. are added as charged substances, and α-tocopherol etc. are added as antioxidants to form membrane components. A substance may also be formed. The ratio of the components of such ribosome membrane component substances is not limited in any way, but preferably sterols are about 0 to 1 part by weight and charged substances are about 0 to 0.2 parts by weight to 1 part by weight of lipid. It is common to add a certain amount.

The volatile organic solvent is preferably one that can dissolve the ribosome membrane component substance and has a low boiling point, and specific examples include chloroform, trichloroethane, acetone, and ethanol. These volatile a! ! The solvents may be used alone or in combination, and when used in combination, those that are miscible with each other are preferred. The amount of such an organic solvent to be used with respect to the membrane component material may be at least an amount capable of dissolving the membrane component material, and is usually used in an amount of 5 to 100 parts by weight per 1 part by weight of the membrane component material.

As the aqueous solvent, water, physiological saline, a buffer solution consisting of phosphoric acid, citric acid, etc., an aqueous solution of sugars such as glucose, mannitol, etc., and a mixture thereof can be used. These aqueous solvents are usually used in an amount of 10 to 1 part by weight of the membrane component material.
1000 parts by weight are used.

Next, a specific operating method of the manufacturing method of the present invention will be explained.

First, a ribosome membrane component substance is dissolved in a volatile organic solvent. In general, membrane component substances can be dissolved in volatile organic solvents relatively easily, but it is more efficient if means such as heating and stirring are used.

A homogeneous mixture can be obtained by subjecting the obtained solution to JIB drying using a double-antibody type, disk type, etc. spray drying device, preferably a solvent recovery type. A homogeneous mixture of substances,
Obtained in powder, oil, and semi-solid form. In general, a homogeneous mixture obtained in powder form is preferred from the viewpoint of swelling speed, and the particle size is preferably as small as possible. In addition, a substance insoluble in a volatile organic solvent (hereinafter referred to as a core substance) is added to a solution consisting of a membrane component substance and a volatile organic solvent, thoroughly dispersed, and then spray-dried to form a powder with an even smaller particle size. You may get . Especially when the ratio of cholesterol in the ribosomal membrane component used is high or when phospholipids made of unsaturated fatty acids are used as the lipid component of the membrane component material, a homogeneous mixture is often obtained in the form of an oil or semi-solid. . In such cases, it is desirable to add the above-mentioned nuclear material.

There are various types of nuclear substances, but among them, those that can be used in aqueous solvents are preferable from the viewpoint of osmotic pressure of ribosome preparations.Specific examples include sugar alcohols such as mannitol, sorbitol, and multidose; glucose, inositol, etc. ims; disaccharide necks such as saccharose, maltose, and lactose; trisaccharides such as maltotriose; cyclic oligosaccharides such as dichlordextrin; polysaccharides such as dextrin and dextran; amino acids such as glycine, lysine, and sodium glutamate; Examples include inorganic salts such as potassium chloride, sodium chloride, and phosphate; organic salts such as citrate; and polymeric compounds such as polyvinylpyrrolidone and dextran sulfate. These may be used alone or in combination. The nuclear material is usually divided into 1 part by weight of the membrane component material.
2 to 50 parts by weight are used. Further, various particle sizes of the additive substance can be used, but those having a particle size of about 150 μm or less are usually used.

An aqueous solvent is added to the obtained homogeneous mixture of the membrane component material or the mixture of the membrane component material and the core material to swell the membrane component material. At this time, the phase transition temperature of the lipid (hereinafter referred to as TC
It is called. ) If the temperature is increased above 100%, the swelling will proceed more rapidly.

Once sufficiently swollen, the target ribosome suspension 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 it is more efficient to heat the temperature to 70°C or higher. Furthermore, it is desirable to perform bubbling with an inert gas such as nitrogen gas during the swelling, dispersion, and post-dispersion processes.

There are no particular limitations on the substances that can be retained in the ribosomes of the present invention, but examples include anticancer drugs such as adriamycin, cytosin arabinoside, and methotrexate, antibiotics such as alphatericin B, insulin, interferon, and glucoamylase. proteins, polysaccharides such as dextran, DNA, R
Examples include nucleic acids such as NA, vitamins such as vitamin A, and general drugs such as sodium salicylate. These substances are used after being dissolved in an aqueous solvent, and include chlorophyll, gramicidin S, and vitamin A.
It is usually more efficient to mix membrane-compatible drugs such as these in an organic solvent together with membrane component substances.

Mata, in order to increase the retention rate of the drug in ribosomes within the formulation, dissolve the prescribed amount of the drug to be retained in a small amount of aqueous solvent, first add this to the spray-dried powder of the membrane component substance, and then swell. , dispersed, and then diluted by adding an aqueous solvent.

To produce ribosome preparations with small particle sizes, an ultrasonic emulsifying machine, a high-pressure pore forming machine, etc. may be used. Furthermore, in order to make the diameter uniform, it is also possible to control the particle size distribution using an ultrafiltration membrane method, such as a polycarbonate membrane filter.

In this way, ribosomal preparations with uniform particle size that retain drugs can be obtained in large quantities with good reproducibility.

This ribosome preparation may be used as it is, or it may be used after separating and removing the drug not retained in the ribosomes by means such as dialysis, gel filtration, or centrifugation.

<Effects of the Invention> In addition to the above-mentioned points, the present invention is superior to known ribosome preparation methods in the following points.

1) Scale-up is easily possible and ribosome preparations can be manufactured industrially.

2) Since spray drying is used, ribosome membrane component substances are not damaged.

3) Ribosome membrane component material obtained by spray drying,
In some cases, a homogeneous mixed powder of this and the nuclear material can be stored in powder form.

4) A ribosomal preparation with a high drug retention rate can be obtained.

5) Ribosomes can be manufactured using almost any lipid.

6) Volatile organic solvents can be recovered.

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

Example I L-α-dipalmitoylphosphatidylcholine (purity 9
9. Weigh out 2.94g and add chloroform 20g
After clearly dissolving in 0 ml of water, the solvent was removed by spray drying. The equipment used was a two-fluid spray dryer (Parvis Mini Spray GA-31, manufactured by Yamato Scientific Co., Ltd.), with a spray air pressure of 1.0 kg/cm2, a liquid feeding rate of approximately 9 g/min, and a chamber population temperature. was approximately 65°C, and the outlet temperature was approximately 50°C.

58.8 mg of the obtained homogeneous white mixed powder was weighed out, and 4 ml of an O, 14M glucose aqueous solution kept at 60° C. was added thereto to sufficiently swell it. When the mixture was kept at 50-60° C. for 7 nights and thoroughly shaken using a vortex mixer and returned to room temperature, a milky white ribosome suspension retaining glucose was obtained.

Take 0.5ml of this ribosome suspension and dialyze it using a cellophane tube (physiological saline, 1fLX 3 times,
5°C), and glucose not retained in ribosomes was separated and removed. Next, glucose in the ribosome suspension was extracted into the aqueous layer by oil/water partitioning according to a conventional method and quantified, and the retention rate was 36.1 kg.

In addition, when the particle size distribution of the ribosome suspension before dialysis was measured using a quasi-elastic light scattering meter, the weight average was 732.
±4 [i0r+m.

Example 2 Fully hydrogenated purified egg yolk lecithin (more than 99 phospholipids, IV
= 1) 4.0 g, :) Weigh out 0.84 g of restyrol and 0.4 g of dicetyl phosphate, and add 5 g of chloroform.
After clearly dissolving in 0ml, the solvent was removed by 0111 mist drying. The oi fog dryer, atomizing air pressure, liquid feeding rate, chamber population temperature, and outlet temperature were the same as in Example 1.

From the obtained homogeneous white mixed powder, 209.6 mg was weighed and added to it was 0.28M which had been kept warm at 60°C.
When 10 ml of a glucose aqueous solution was added and the same procedure as in Example 1 was carried out, a milky white ribosome suspension retaining glucose was obtained.

0.5 ml of this solution was taken and dialyzed in the same manner as in Example 1 to determine the retention rate, which was found to be 25.3.

Example 3 Completely hydrogenated purified soybean lecithin (phospholipid 9 or more journals, IV-
3) 3.69 g, cholesterol 0.7'Jg and dicetyl phosphate (1,371B) were taken out and clearly dissolved in 50 w1 chloroform, and the solvent was removed by spray drying. Spray drying device, spray air pressure ,
The liquid feeding rate, chamber population temperature, and outlet temperature were the same as in Example 1.

From the obtained homogeneous white mixed powder, 3.67 g was weighed out, and 250 ml of O-packed sodium salicylate physiological saline solution kept at 60°C was added thereto, and the same procedure as in Example 1 was carried out. A milky white ribosomal suspension retaining sodium was obtained.

On this day, 0.5 ml was taken and subjected to gel filtration J (1 cm × 1 B cm, physiological saline) using Sephadex G-50 to separate and remove sodium salicylate that was not retained by ribosomes. Next, sodium salicylate in the ribosomal fraction was extracted into the aqueous layer by oil/water partitioning according to a conventional method and quantified, and the retention rate was 14.2%.

Example 4 Fully hydrogenated purified egg yolk lecithin 3.2g, cholesterol 0
．． 67 g and 0.32 g of dicetyl phosphate were weighed out, clearly dissolved in 200 ml of chloroform, and the solvent was removed by spray drying. 6-song spray dryer, atomizing air pressure, liquid feeding rate, chamber population temperature, outlet temperature. was the same as in Example 1.

94.2B was weighed out from the obtained homogeneous white mixed powder, and in the same manner as in Example 1, 1 was added instead of the glucose aqueous solution.
When prepared using 8+ul of dextran T40 physiological saline solution, a milky white ribosome suspension retaining dextran T40 was obtained.

Is this ribosome cloudy? & 1 ml and gel filtration using Sepharose CL-48 (2.2 cm x
42 cm, physiological saline), and dextran T40 that was not retained in ribosomes was separated and removed. Next, the ribosomal fraction dextran T40 was mixed with oil/T40 according to a conventional method.
When extracted into the aqueous layer by water partition and quantitatively determined, the retention rate was 9.1! It was.

Example 5 5.27 g of fully hydrogenated purified soybean lecithin, 0.37 g of dicetyl phosphate, and 0.3 g of DL-α-tocopherol.
80 g was weighed out, clearly dissolved in 50 ml of chloroform, cooled, and the solvent was removed by spray drying. Spray drying equipment, spray air pressure, liquid delivery speed, chamber population temperature,
The outlet temperature was the same as in Example 1.

89.1 mg was weighed out from the obtained homogeneous white mixed powder and prepared in the same manner as in Example 1 using 10 ml of physiological saline, resulting in milky white ribosomes retaining DL-α-tocopherol in the thimble membrane. A suspension was obtained.

Example 6 Fully hydrogenated purified egg yolk lecithin 40g, cholesterol 8.
4 g and 1-4 g of dicetyl phosphate were weighed out, clearly dissolved in 500 ml of chloroform, and the solvent was removed by spray drying. The spray drying device, spray air pressure, liquid feeding rate, chamber population temperature, and outlet temperature were the same as in Example 1.

Weighed 20.96 mg of the homogeneous white mixed powder obtained, and added 0.28M8 which had been kept at 60°C in advance.
M Glucose solution was added once and the procedure was carried out in the same manner as in Example 1 using an Ajihomo mixer. A milky white ribosome suspension containing glucose was obtained.

0.5 ml of this solution was taken and dialyzed in the same manner as in Example 1 to determine the retention rate, which was 32,096.

In addition, when the particle size distribution of the ribosome suspension before dialysis was measured using a quasi-elastic light scattering meter, the weight average was 847±
It was 591 nm.

Example 7 Shi-α-dipalmitoylphosphatidylcholine 2.94
g was weighed out and dissolved clearly in 100 ml of chloroform, and then 10.2 g of D-mannitol, which had been previously sieved through a 100-mesh sieve, was dispersed in this chloroform solution.
Suspended. While thoroughly stirring this suspension with a stirrer, the solvent was removed by spray drying. Spray drying equipment,
The atomizing air pressure, liquid feeding rate, chamber population temperature, and outlet temperature were the same as in Example 1.

262.8B was weighed out from the homogeneous tt white mixed powder obtained, and 4 liters of distilled water for injection, which had been kept at 60°C in advance, was added to it.
ml was added and the mixture was sufficiently swollen. Temperature 50-60℃
The mixture was thoroughly shaken using a portic mixer while the temperature was maintained at room temperature, and when the temperature was returned to room temperature, a milky white ribosome suspension was obtained.

When the particle size distribution of this ribosome suspension was measured using a quasi-elastic light scattering meter, the weight average was 803±408 nm.
There was.

Example 8 Fully hydrogenated purified egg yolk lecithin 3.2g, cholesterol 0.67g and dicetyl phosphate 0.32. After 100 ml of chloroform was weighed out, 10.2 g of D-mannitol, which had been passed through a 100-mesh sieve, was dispersed and suspended in the chloroform solution. As in Example 7, the solvent was removed by spray drying while stirring with a stirrer to obtain a homogeneous white mixed powder. The spray drying device, spray air pressure, liquid feeding rate, chamber population temperature, and outlet temperature were the same as in Example 1.

141.3 mg of the obtained homogeneous white mixed powder was weighed out, 4 ml of 0.14M glucose aqueous solution kept at 60°C was added thereto, and the same procedure as in Example 1 was carried out. A ribosome suspension was obtained.

When the glucose retention rate was determined in the same manner as in Example 1, it was 24.8 zero.

Example 9 The same procedure as in Example 8 was carried out using 3.2 g of fully hydrogenated purified soybean lecithin, 10.2 g of O-mannitol sieved through a 100-mesh sieve, 0.67 g of cholesterol, 1.16 g of stearylamine, and 100 ml of chloroform. , a homogeneous white mixed powder was obtained. The spray drying device, spray air pressure, liquid feeding speed, chamber inlet temperature, and outlet temperature were the same as in Example 1.

711.5 mg was weighed out from the obtained homogeneous white mixed powder, and to this was added O, 14M glucose aqueous solution IQml which had been kept at 50°C in advance, and the following procedure was carried out in the same manner as in Example 7. A ribosome suspension was obtained.

When the glucose retention rate was determined in the same manner as in Example 1, it was 24.8 zero.

Example 10 L-α-dipalmitoylphosphatidylcholine 2.94
Weigh out 0.67 g of cholesterol and 0.32 g of dicetyl phosphate, and add 100 ml of chloroform.
After clearly dissolving in chloroform, 102 g of D-sorbitol, which had been sieved through a 100-mesh sieve, was dispersed and suspended in chloroform. While thoroughly stirring this suspension with a stirrer, the solvent was removed by spray drying. Spray drying equipment, 9 mist air pressure, liquid feeding rate, chamber population temperature,
The outlet temperature was the same as in Example 1.

A sample of 282.6 mg of the homogeneous white mixed powder obtained was sampled, and 4 ml of 1* Dextran T40 aqueous solution kept at 60°C was used in the same manner as in Example 1. As a result, Dextran T40 was retained. A milky white ribosome suspension was obtained. When the retention rate was determined in the same manner as in Example 4, it was found to be 8396.

Example 11 Fully hydrogenated purified egg yolk lecithin 32g, cholesterol 6.
Weigh out 7 g and 1.6 g of stearylamine, dissolve them clearly in 500 ml of chloroform, and add 1.6 g of stearylamine in advance.
102 g of D-mannitol passed through a 00 mesh sieve was dispersed and suspended in this chloroform solution. While thoroughly stirring this suspension with a stirrer, the solvent was removed by spray drying. Spray drying equipment, atomizing air pressure, liquid feeding speed,
The chamber population temperature and outlet temperature were the same as in Example 1.

35.6 g of the obtained white mixed powder was weighed out, and a 0.14 M glucose aqueous solution Ik kept at 60°C was added thereto. The procedure was carried out in the same manner as in Example 6. As a result, glucose was retained. A milky white ribosome suspension was obtained. When the glucose retention rate was determined in the same manner as in Example 1, it was found to be 29.1! It was t.

When the particle size distribution of this ribosome suspension was measured using a quasi-elastic light scattering meter, the weight average was 768±512 nm.
Met.

Claims (1)

[Claims] A method for producing ribosomes, which comprises spray-drying a solution of ribosome membrane component substances dissolved in a volatile organic solvent to prepare a homogeneous mixture, and then dispersing the homogeneous mixture in an aqueous solvent.