JPH06329533A - Production of lilposome preparation - Google Patents

Abstract

PURPOSE:To provide a method for incorporating compounds having free or salt-form carboxyl groups and amino groups in the molecule into liposome simply with no use of special tools. CONSTITUTION:An aqueous solution of a compounds having free or salt-form carboxyl and amino groups is added little by little to phospholipid with stirring, for example, it is added or poured in an amount of 0.01 to 2ml in 5 to 10 portions based on 1g. of phospholipid, kneaded, then diluted with an aqueous solvent such as distilled water, sodium chloride aqueous solution, aqueous glucose, or buffer solution to give the objective liposome preparation. As a phospholipid, is suitable a combination of phosphatidyl-choline and phosphatidic acid and, when needed, a sterol is added as a membrane stabilizer and tocopherol is added as an antioxidant. The compound to be included is, for example, L- leucovorin, glycylglycine, glutathione, or mitomycin derivative having free or salt-forming carboxyl groups and amino groups in the molecule.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily encapsulating a compound having a carboxyl group and an amino group in a molecule, which is free or has formed a salt, into a liposome with high encapsulation efficiency.

[0002]

2. Description of the Related Art In the field of medicine, liposome preparation is one of the means for enhancing the therapeutic effect of drugs. A method for encapsulating a water-soluble drug in liposomes has been hitherto described in Bangham.
[Journal of Molecular Biology (J.
Mol.Biol.), 13 , 238 (1965)], liposome preparation method, ethanol injection method [J. Cell Biol., 66 , 621 (1975)], French press method. [FEBS Lett., 99 , 210
(1979)], freeze-thaw method [Archives of Biochemistry and Biophysics (Arch. Bichem.
Biophys.), 212, 186 (1981)], reverse phase evaporation method [Proceeding of the National Academy of
Sciences USA (Proc.Natl.Acad.Sc
i.USA), 75 , 4194 (1978)] is known.

However, the method of Bangham et al. Has problems that the particle size distribution is wide and the encapsulation rate of the substance in the aqueous phase of the liposome is low. Is difficult to remove. The French press method requires a special device called the French press, and the freeze-thaw method,
There are problems that the distribution of particle diameters is wide, and that retention efficiency is lowered because sugar, high ionic strength, divalent cation, and high concentration electrolyte are used. In the reverse phase evaporation method, a flammable organic solvent such as ether must be used, which is problematic in terms of operation safety. Further, in the method of Bangham et al. And the freeze-thaw method, an aqueous solution of a drug is added all at once and a liposome is prepared without kneading.

[0004]

The object of the present invention is to use a liposome preparation containing a compound having a carboxyl group and an amino group in the molecule, which is free or has formed a salt, in a liposome, using a simple and special device. It is to provide a method of manufacturing with a high encapsulation rate without performing the above.

[0005]

Means for Solving the Problems In the present invention, an aqueous solution of a compound having a carboxyl group and an amino group in a molecule, which is free or forms a salt, is added to a phospholipid little by little while stirring, and after kneading, an aqueous solvent is added. The present invention relates to a method for producing a liposome preparation, which comprises diluting with. The compound to be encapsulated in the liposome by the method of the present invention may be any compound having a carboxyl group and an amino group in the molecule which are free or form a salt, and examples thereof include L-leucovorin represented by the following structural formula: Glycylglycine, glutathione, mitomycin (MM) having a free or salt-forming carboxyl group and amino group in the molecule
Examples thereof include C and D derivatives.

[0006]

[Chemical 1]

The phospholipid used in the method of the present invention may be used as it is, or a thin film obtained by dissolving the solvent in a solvent such as chloroform and then distilling off the solvent may be used. Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, and other natural or synthetic phospholipids, hydrogenated phospholipids or glyceroglycolipids. They may be used alone or in combination, but a combination of phosphatidylcholine and phosphatidic acid is preferably used.

In the method of the present invention, a compound having a free or salt-forming carboxyl group and an amino group in the molecule is added little by little to a phospholipid with stirring and kneading is carried out, for example, by adding a phospholipid. Glass beads were placed in the eggplant-shaped flask that was placed, and an aqueous solution of a compound was added or dropped in 0.01 to 2 ml per 1 g of phospholipid in 5 to 10 times, and kneaded with stirring, and phospholipid was placed. An example is a method in which an aqueous solution of the compound is added or dropped in 0.01 to 2 ml per 1 g of phospholipid in 5 to 10 times in a mortar or the like, and kneaded while stirring with a pestle or the like.

The concentration of the aqueous solution of the compound added to the phospholipid is not particularly limited as long as the compound does not precipitate because it changes depending on the solubility of the compound used in water, but the higher the concentration, the higher the encapsulation rate. The amount of the aqueous solution of the compound used is 0.1 to 1 per 1 g of phospholipid.
It is 0 ml, preferably 0.5 to 2 ml. If necessary, sterols such as cholesterol may be used as a membrane stabilizer, tocopherol may be used as an antioxidant, and stearylamine, dicetyl phosphate, ganglioside may be used as a charged substance, if necessary.

An aqueous solution of the compound to be encapsulated is added little by little to the phospholipid while stirring and kneading, and then an aqueous solvent is added to form liposomes. As the aqueous solvent to be added, for example, distilled water, saline, an aqueous glucose solution or a buffer such as a phosphate buffer or Tris-hydrochloric acid buffer is 1 g of phospholipid.
10 to 1000 ml, preferably 20 to 500 m
used.

Further, substances that constitute buffer solutions such as phosphoric acid, acetic acid and citric acid, isotonic agents such as sodium chloride, glucose and glycerin, antioxidants such as ascorbic acid and glutathione, chlorbutanol, paraben and the like. These additives can be made to coexist mainly in the aqueous layer of the liposome by using a preservative or the like together with the encapsulating compound or with the aqueous solvent.

The compound not encapsulated in the liposome in the method of the present invention can be removed by means such as centrifugation or various chromatographies. Also, the hole diameter is 100n
Extruder (Lipex Biomembranes) using m polycarbonate film
The particle size of the liposome can be adjusted by sizing according to the method manufactured by the company.

Although the liposome preparation obtained by the method of the present invention can be used as it is, it may be lyophilized by adding an excipient such as mannitol, lactose or glycine depending on the purpose of use or storage. The liposome preparation obtained by the method of the present invention is generally used as an injection, but oral, nasal, eye, transdermal, suppository,
It can also be processed and used as an inhalant.

Examples, reference examples and test examples of the present invention will be shown below. In the following examples and reference examples,
Compound A and Compound 1 which are mitomycin derivatives
The structure of 8 is as follows.

[0015]

[Chemical 2]

[0016]

【Example】

Example 1 Soybean phosphatidylcholine (45 mg), phosphatidic acid (5 mg), and glass beads having a diameter of 7 mm (about 6 g) were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. To the obtained lipid thin film, 50 μl of an aqueous glutathione solution (200 mg / ml) was added in 10 μl portions in 5 batches, and the mixture was well kneaded. Then, distilled water was added to 0.
45 ml was added and stirred to form liposomes.

Example 2 45 mg of soybean phosphatidylcholine, 5 mg of phosphatidic acid and about 6 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. To the obtained lipid thin film, 50 μl of an aqueous glycylglycine solution (86 mg / ml) was added in 10 μl portions in 5 batches, and well kneaded with stirring. Next, 0.45 ml of 2.2% saline was added and stirred to form liposomes.

Example 3 45 mg of soybean phosphatidylcholine, 5 mg of phosphatidic acid and about 6 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound A [Chemical and Pharmaceutical Bulletin (Che
m. Pharm. Bull.) , 37, 1128 (1989)] (100 mg /
50 μl of an aqueous solution containing 10 ml of water was added in 5 batches of 10 μl each, and the mixture was well kneaded. Next, 0.45 ml of 0.5% saline was added and stirred to form liposomes.

Example 4 270 mg of soybean phosphatidylcholine, 30 mg of phosphatidic acid, and about 6 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (5 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound A (4
0.01M phosphate buffer (pH 0mg / ml) (pH
7.0) 0.5 ml was added in 0.1 ml portions in 5 portions, and the mixture was well kneaded with stirring. Then, phosphate buffer 9.5m
1 was added and stirred to form liposomes.

Example 5 Soybean phosphatidylcholine (45 mg), phosphatidic acid (5 mg), and glass beads having a diameter of 7 mm (about 6 g) were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. The obtained lipid thin film contained Compound 1 (JP-A-60-255789) (100 mg / ml) in an amount of 0.1.
10 μl of 50 μl of 05M phosphate buffer (pH 7.0)
Each of them was added in 5 batches and kneaded well. Then
0.45 ml of 0.05 M phosphate buffer (pH 7.0) was added and stirred to form liposomes.

Example 6 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 2 is added to the obtained lipid thin film.
(JP-A-60-255789) (25 mg / ml of 0.05 M phosphate buffer (pH 7.0) containing 35 mg / ml)
Each μl was added in 5 batches and well kneaded. Next, 0.05M phosphate buffer (pH 7.0) 0.225
ml was added and stirred to form liposomes.

Example 7 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 3 is added to the obtained lipid thin film.
(JP-A-60-255789) (100 mg / ml), 25 μl of 0.05 M phosphate buffer (pH 7.0) was added in 5 μl portions in 5 batches, and the mixture was well kneaded. Next, 0.05M phosphate buffer (pH 7.0) 0.225
ml was added and stirred to form liposomes.

Example 8 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 4 is added to the obtained lipid thin film.
(WO88 / 01622) (12 mg / ml) containing 5 μl of 0.05 M phosphate buffer (pH 7.0)
Each μl was added in 5 batches and well kneaded. Next, 0.05M phosphate buffer (pH 7.0) 0.225
ml was added and stirred to form liposomes.

Example 9 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 5 was added to the obtained lipid thin film.
25 μl of 0.05M phosphate buffer (pH 7) containing (WO88 / 01622) (30 mg / ml) was added in 5 μl portions in 5 batches, and well kneaded with stirring. Then 0.
0.225 ml of 05M phosphate buffer (pH 7.0) was added and stirred to form liposomes.

Example 10 Soybean phosphatidylcholine 900 mg, phosphatidic acid 150 mg, cholesterol 250 mg, diameter 7 mm
Approximately 25 g of the glass beads of 1 was placed in an eggplant-shaped flask. After adding 2 ml of chloroform and stirring to dissolve the lipid,
Chloroform was distilled off with a rotary evaporator.
1 ml of 0.01 M Tris-hydrochloric acid buffer solution (pH 7.0) containing Compound A (100 mg / ml) in the obtained lipid thin film
Was added in 200 μl portions in 5 batches and kneaded well. Then 0.01 M Tris containing 4.9 g lactose-
49 ml of hydrochloric acid buffer solution (pH 7.0) was added and stirred to form liposomes.

Example 11 Soybean phosphatidylcholine (45 mg), phosphatidic acid (5 mg), and glass beads having a diameter of 7 mm (about 6 g) were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. To the obtained lipid thin film, 50 μl of L-leucovorin aqueous solution (100 mg / ml) was added in an amount of 10 μl each.
The mixture was added in portions and kneaded well. Next, 0.45 ml of distilled water was added and stirred to form liposomes.

Example 12 Cation-exchange resin (Dowex 50W-Xa, 50-100 mesh) prepared from the liposomes obtained in Example 11
Free compound A was removed using 40 g. Dispense 2 ml each of the obtained liposome suspension into a glass vial,
Lyophilization was performed to obtain a liposome preparation.

Comparative Example 1 Soybean phosphatidylcholine (45 mg) and phosphatidic acid (5 mg) were placed in an eggplant-shaped flask. 2 ml of chloroform
Was added, and the mixture was stirred to dissolve the lipid, and then chloroform was distilled off with a rotary evaporator. 0.5 ml of glutathione (20 mg / ml) was added to the obtained lipid thin film at once, and liposomes were formed without kneading the lipid and the drug.

Comparative Example 2 270 mg of soybean phosphatidylcholine and 30 mg of phosphatidic acid were placed in an eggplant-shaped flask. Chloroform 5
After adding ml and stirring to dissolve the lipid, chloroform was distilled off with a rotary evaporator. To the obtained lipid thin film, 10 ml of 0.01 M phosphate buffer (pH 7.0) containing compound A (2 mg / ml) was added at once, and liposomes were formed without kneading the lipid and the drug.

Comparative Example 3 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 6 (WO88 / 016) having no carboxyl group in the molecule in the obtained lipid thin film
No. 22) (15 mg / ml) in 0.05 M phosphate buffer (pH 7.0) (25 μl) was added in 5 μl portions in 5 batches, and the mixture was well stirred and kneaded. Next, 0.225 ml of 0.05 M phosphate buffer (pH 7.0) was added and stirred to form liposomes.

Comparative Example 4 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid, and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 7 (WO88 / 016) having no carboxyl group in the obtained lipid thin film
22) (100 mg / ml) in 0.05 M phosphate buffer (pH 7.0) (25 μl) was added in 5 μl portions in 5 batches, and the mixture was well kneaded with stirring. Next, 0.225 ml of 0.05 M phosphate buffer (pH 7.0) was added and stirred to form liposomes.

Comparative Example 5 22.5 mg of soybean phosphatidylcholine, 2.5 mg of phosphatidic acid and about 3 g of glass beads having a diameter of 7 mm were placed in an eggplant-shaped flask. Chloroform (2 ml) was added, the mixture was stirred to dissolve the lipid, and then the chloroform was distilled off with a rotary evaporator. Compound 8 (WO88 / 016) having no carboxyl group in the obtained lipid thin film
22) (50 mg / ml) in 0.05 M phosphate buffer (pH 7.0) (25 μl) was added in 5 μl portions in 5 batches, and the mixture was well stirred and kneaded. Next, 0.225 ml of 0.05 M phosphate buffer (pH 7.0) was added and stirred to form liposomes.

Test Example The encapsulation rate of the compounds of Examples and Comparative Examples in liposomes was determined as follows. Examples 1-11 and Comparative Examples 1-
The free compound and the liposome were separated from the liposome obtained in 5 by gel filtration (1.5 × 15 cm) using Sepharose CL-6B. After 5% Triton X-100 was added to the liposome fraction to break the liposome, high performance liquid chromatography [HPLC; YMC pack AM-312 ODS, 6 ×] was used.
150 mm, mobile phase 0.05 M phosphate buffer (pH 7.0): acetonitrile: methanol (75: 17: 8), flow rate 1.2 ml / min, detection wavelength 254 nm] The amount of compound encapsulated in the liposome and the amount of free compound Was quantified and the encapsulation rate was calculated by the following formula. The results are shown in Table 1.

[0034]

[Equation 1]

[0035]

[Table 1]

As shown in Table 1, the liposome preparation obtained by the method of the present invention showed a higher encapsulation rate than the preparation in which the lipid and the drug were not kneaded (Comparative Examples 1 and 2). In addition, when the drug to be encapsulated is a compound having neither a free or salt-forming carboxyl group nor an amino group in the molecule, a case where it is a compound having a free or salt-forming carboxyl group or an amino group in the molecule It did not show a high inclusion rate in comparison.

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for easily encapsulating a compound having a carboxyl group and an amino group, which is free or has formed a salt in a molecule, in a liposome at a high encapsulation rate.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuno Kono 1504-2, Shimochikari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture (72) Inventor Masatsugu Kasai 3-12-15 Matsugaoka, Kugenuma, Fujisawa-shi, Kanagawa

Claims (2)

[Claims] 1. An aqueous solution of a compound having a carboxyl group and an amino group in a molecule, which is free or has formed a salt, is added little by little to a phospholipid while stirring, and after kneading, it is diluted with an aqueous solvent. Method for producing liposome preparation. 2. A compound having a free or salt-forming carboxyl group and an amino group in the molecule has L-leucovorin, glycylglycine, glutathione, and a free or salt-forming carboxyl group and an amino group in the molecule. The method for producing a liposome preparation according to claim 1, which is selected from mitomycin derivatives.