JPS63201117A - Production of liposome - Google Patents

Abstract

PURPOSE:To obtain a liposome free from residual organic solvent, preventing the degradation of active drug, by preparing a liposome having a membrane composed of a (hydrogenated) phospholipid, adding and fusing Ca<2+> thereto and adding a drug component and a sequestering agent to remove the Ca<2+> ion. CONSTITUTION:A phospholipid and/or a hydrogenated phospholipid, preferably a hydrogenated phospholipid having an iodine value of <=30 is used as a membrane material and a liposome is prepared by ultrasonic process, etc. The liposome is fused by adding calcium ion and stirring the mixture. The produced mixture is added with a drug to be included, thoroughly stirred and added with a sequestering agent (e.g. EDTA) to remove the calcium ion and obtain the objective liposome.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to an improved method for producing drug-loaded liposome formulations.

(Prior art) Liposomes are known as multilamellar liposomes (MLV HMultlla+el) due to their morphology.
lar VeSi-cle), small unilamellar liposomes (SUV; Small Unilamellar V
esicle), large unilamellar liposomes (LIJ
V; Large 1Jnfla*ellar Ve
sicla).

Among them, the manufacturing method of LUV that can retain macromolecules such as proteins and has good retention efficiency is as follows: ■ Prepare SUV of acidic phospholipid, then add Ca2+ and fuse it to form a potworm-shaped cylinder, and finally EDT
Dividing Ca'+ by A (Ca'+ fusion method.

■ 55-60 ether solution containing lipids containing drugs
An ether injection method in which when injected into an aqueous solution at ℃, the injected ether comes into contact with water, turns into steam, and forms LUV while rising to the surface of the aqueous solution.

■SUV with structural defects caused by ultrasonication of MLV at temperatures below Tc
An annealing method in which a compound is formed and then incubated at a temperature higher than Tc to fuse.

■A freeze-thaw fusion method in which an SUV manufactured using the ultrasonic method is first frozen, then returned to the greenhouse to thaw, and then lightly treated with ultrasonic waves again.

■ Add an aqueous solution to the n-hexane solution of phospholipid and 5pan 80, homogenize it to make a W10 emulsion, distill off the n-hexane under reduced pressure to make a W/L mixture,
W10/W emulsion carbon method in which an aqueous solution containing a highly hydrophilic surfactant is added and homogenized to form W/L/W, and finally excess surfactant is removed by dialysis or the like.

■Add an aqueous solution to an ether solution of lipids and apply ultrasound treatment.
Make W10 Emaru Gene. Next, the ether is distilled off under reduced pressure to form a gel, and after shaking gently, the remaining organic solvent is distilled off again under reduced pressure.This is a reverse phase evaporation method.

etc. are known.

(Problems to be Solved by the Invention) However, harsh manufacturing methods such as heating and ultrasonication are included in liposomes.

If the medicinal substance is a substance such as a protein or peptide, this may lead to partial decomposition, and if an organic solvent is used, the removal of this solvent from the formed liposomal confectionery may be incomplete. It had such drawbacks.

In view of these circumstances, the present inventors have made extensive studies and found that after fusing liposomes whose membrane material is phospholipids and/or hydrogenated phospholipids with calcium ions, adding the drug encapsulated therein, The present inventors have discovered that by removing calcium ions using a sequestering agent, liposomes can be obtained without altering the quality of the drug contained in the liposomes, and in which no organic solvents remain, leading to the completion of the present invention.

(Means for Solving the Problems) The present invention relates to an improvement of the Ca'+ fusion method, and the conventional method has the above-mentioned drawbacks because the drug is added from the beginning and heating, ultrasonic treatment, etc. Was.

In the present invention, the membrane material is a phospholipid and/or a hydrogenated phospholipid, preferably a hydrogenated phospholipid with a wax value of 30 or less, and a liposome is prepared by ultrasonication or the like, and then calcium ions are added and stirred. and fuse the liposomes.

Add the water-soluble drug to be encapsulated in the liposomes and stir thoroughly. Then EDTA is added to remove calcium ions and obtain LUV.

(Effect) The liposome preparation prepared by the method of the present invention has a high uptake rate and does not require harsh manufacturing methods such as heating or ultrasonic treatment, so the medicinal substance contained in the liposome does not undergo decomposition.

Furthermore, if a method that does not use an organic solvent is selected when the liposome is first treated with GIII, no organic solvent will remain in the liposome.

Next, examples of the present invention will be shown. The blending ratios are in weight %.

Example 1 Table 1゜ ■ Hydrogenated lecithin 0.50 ■ Purified water 9B, 69 ■ L-ascorbic acid 0.20 ■ Potassium hydroxide 0.01 ■ Calcium chloride 0. IO ■Disodium edetate 0.50 Among the components (2), 0.5 parts by weight and the component (2) are heated and dissolved at 70°C to form a liquid crystal.

Next, the remaining component (2) is added and shaken and stirred to disperse the liquid crystal to obtain a liposome solution, which is treated with ultrasonic homogenization until it becomes transparent to obtain MLV.

After that, after adding the component ■ and fusing the MLV, ■■
After adding the ingredients (2) and stirring thoroughly, add the ingredients (2) to obtain LUV.

Comparison N1 0.5 parts by weight of the component (1) and the component (2) were heated and dissolved at 70°C to form a second crystal.

Next, the remaining components (■) and (■■) are added and shaken and stirred to disperse the liquid crystal to obtain a liposome solution, which is treated with an ultrasonic homogenizer until it becomes transparent to obtain ML.
Get V. Add the component ■ and fuse the MLV,
Furthermore, add the component (■) to obtain LUV.

The LUV@ suspension obtained by the above method was placed in a υCC cellophane-chave dialysis pack and dialyzed against isotonic phosphate buffer (5°C, 11 x 1 hour x 6 times) to retain it within the liposome. Ascorbic acid that was not present was separated and removed. Table 1 shows the ascorbic acid uptake rates of the liposomes of Examples and Comparative Examples.

The uptake rate was expressed in terms of the amount of drug used. The amount of drug contained in the liposomes was determined by destroying the drug with a mixture of ether:methanol (5:1) and releasing the ascorbic acid using the Japanese Pharmacopoeia (10th revision).
C-35), 0. Titration was performed using IN iodine solution.

Furthermore, in Comparative Example 1, remarkable browning and off-odor were observed during the preparation of MLV, but no abnormality was observed in Examples.

Example 2 ■Concentrated glycerin 3.00 ■Hydrogenated lecithin 0.80 ■Purified water 95.10 ■Adenosine triphosphate 0.50 ■Calcium chloride 0.10 ■Edetate disodium 0.50 ■Ingredients were heated at 90°C. The mixture was heated to , and the component (2) was added thereto and stirred to uniformly swell. The mixture was further stirred on an 80°C water bath until it became a white homogeneous paste. 80℃ for this
The heated ingredients (2) were added to the mixture, and the paste was left at 80°C for 3 minutes to form a paste. Next, the mixture was stirred for 3 minutes using a homomixer while maintaining the temperature at 80°C, and then cooled to room temperature to obtain SUv.

Add the component (2) to the SUV suspension, fuse the liposomes, add the component (2), stir thoroughly, add the component (2), remove calcium ions, and obtain LUV.

Comparative Example 2 The component (1) was heated to 90°C, and the component (2) was added thereto and stirred to uniformly swell. The mixture was further stirred on an 80°C water bath until it became a white homogeneous paste. On the other hand, the component (2) was heated to 80°C, the component (2) was added, stirred and dissolved, and added to the former, and the paste was allowed to stand at 80°C for 3 minutes to expand the paste.

Next, the mixture was stirred for 3 minutes using a homomixer while being maintained at 80°C, and then cooled to room temperature to obtain SUv.

- Add component (2) to the 5uvs suspension to fuse the liposomes, then add component (2) to remove calcium ions to obtain LUV.

The LUV suspension obtained by the above method was washed twice by adding an isotonic phosphate buffer and using ultracentrifugation (85,000 x g for 30 minutes).

Table 2 shows the adenosine triphosphate (ATP) uptake rates of the liposomes of Example 2 and Comparative Example 2.

The amount of drug contained in the liposome was calculated from the absorbance at 260 nm after destruction with an ether:methanol mixture (5:1).

Table 2

Claims (1)

[Claims] After preparing liposomes whose membrane material is phospholipids and/or hydrogenated phospholipids, calcium ions are added to fuse the liposomes, and calcium ions are removed by adding an encapsulated drug and a sequestering agent. A method for producing liposomes, which comprises reconstituting liposomes.