JPS5976021A - Liposome pharmaceutical containing substance fk-565 - Google Patents

Abstract

PURPOSE:The titled pharmaceutical, containing a substance FK-565, and capable of enhancing further the physiological activity. CONSTITUTION:A pharmaceutical, containing liposome, and prepared by dissolving a natural phospholipid, e.g. egg yolk lecithin, soybean lecithin or sphingomyelin or phosphatidyl serine, or a synthetic phospholipid, e.g. distearoylphosphatidyl choline or dipalmitoylphosphatidyl choline, capable of forming a liposome membrane, in an organic solvent, e.g. chloroform, introducing the resultant solution into a suitable vessel, distilling away the solvent under reduced pressure, forming a thin membrane on the inner surface of the vessel, introducing an aqueous solution of a substance FK-565 thereinto, and shaking or treating the resultant mixture with ultrasonic waves to give a suspension of the liposome (fine particles). The resultant liposome can be isolated by means of centrifugation, etc. The substance FK-565 has the antitumor and phylactic action.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to liposome formulations containing the FK-565 substance.

It is known that the compound represented by
No. 449).

FK-565 substance has certain antitumor effects and anti-infective effects even when it is dissolved in distilled water for injection and administered as an injection, or when it is orally administered as a solid preparation such as a tablet, granule, or capsule. Although it has a protective effect,
This invention was made for the purpose of further enhancing these physiological activities.

The FK-565 substance-containing liposome of the present invention has a structure in which the FK-565 substance is encapsulated in a film made of phospholipid or the like.

Examples of phospholipids that form lysome membranes include natural phospholipids such as egg yolk lecithin, soybean lecithin, sphingomyelin, phosphatidylserine, phosphatidylglycerol, phosph-7tidyl isositol, dihonuphatidylglycerol, and phosphatidylethanolamine, and distearoylphosphatidylcholine. , dipalmitoylphosphatidylcholine, dipalmitoylphosphadusylethanolamine, and other synthetic phospholipids. Conventional additives such as cholesterol, charged lipids such as dicetyl phosphate, phosphatidic acid, and stearylamine, or α-tocopherol may be appropriately added to these phospholipids.

The PK-565 substance-containing liposome of this invention can be manufactured by a conventional manufacturing method.

That is, the liposome film-forming substance as described above is dissolved in an organic solvent such as chloroform or ethanol, placed in a suitable container, and the solvent is distilled off under reduced pressure to form a thin film on the inner surface of the container. Obtain a suspension of (somesomes). In addition to water, solvents for preparing the aqueous solution of the FK-565 substance include physiological saline, buffers such as phosphate buffers and tris-aminomethane buffers, aqueous solutions of glucose and sorbitol, or mixtures thereof. Liquid may also be used.

In addition, the liposome of the present invention can be prepared by adding an aqueous solution of the FK-565 substance to a solution of the liposome membrane-forming substance dissolved in an organic solvent such as ethyl ether or diisopropyl ether, and emulsifying the substance by ultrasonic treatment, etc.
It can also be produced by distilling off the organic solvent under reduced pressure.

In addition to the surfactant removal method (Detergent r
emova engineering), ether injection method (Ether 1nj)
The liposome of the present invention can be produced by any method, and the method for producing the liposome is not particularly limited.

The liposomes thus produced are isolated by conventional means such as centrifugation. The single-barrel liposome is purified by redispersing it in an aqueous solvent for dissolving the FK-565 substance and then centrifuging it.

The liposome thus obtained is used by suspending it in an appropriate solvent, or by lyophilizing it and redispersing it in an appropriate solvent.

As is clear from the following test results, the FK-565 substance-containing liposome of the present invention has excellent anti-tumor and infection-preventing effects.

Test Example 1: P388 liver metastasis inhibitory effect Test method: Bearer animals (DBA/2
P2S5 (Maunu lymphocytic leukemia cells) was injected into mice 1x.
1 week later, peritoneal cells (P2S5) were collected from the mouse using a 5 g/volume syringe, Hank's solution was added, and the cells were centrifuged twice (1000 rpm).
m.

5 minutes). Suspend this in Hank's solution, 5XID/
g/! A cell suspension was prepared. This cell suspension 0.1
Shoulders were transplanted from the tail vein of DEA/2 mice (female, 8 weeks old, 5 mice per group) (5 x 104/mouse). The drug is administered into the tail vein on the 1st molar and 3rd day after transplantation. (0,1 shoulder//mouse, once a day). Seven days after transplantation, each mouse is exsanguinated and the liver is removed into Hank's solution. The number of P388 colonies appearing on the surface of the liver swelling is measured under a stereoscopic microscope.

Test results: *P<,0.05 **P<0.01 ***P<0.001 Test example 2: Effect on P388 and P815 solid cancer Test method: In the same manner as Test example 1, P2S5 and P815 (mouse mastocytoma) cell suspension (2X10”/
I made a stomach/). 0.05 m/ml of this cell suspension was transplanted into DBA/2 mice (8 weeks old, 7 mice per group) (IX104/mouse). The drug was administered subcutaneously 6 days before transplantation and 4 days after transplantation (0.2 m/day), respectively.
/mouse, once a day) after transplantation 1 intimation stitch (P815) and 11 days later (P388
), measure the major axis and minor axis of one sore using a caliper,
The volume of the tumor was determined using the following formula.

Volume (original') = 0.4 x major axis - x minor axis - 2 Test results: (Effect on P388 solid tumor) *P<0.05 [Effect on P815 solid tumor] * P<0.05 ** P<0 .0l -7= Test Example 3: Infection protection against Candida albicans test method: Each drug is administered into the tail vein of ddY mice (female, 4 weeks old, 6 mice per group). One day after administration, Candida albicannu dust 65 was cultured in Sabouraud liquid medium at 30°C for 148 hours, centrifuged to collect the bacteria (600 rpm, 20 minutes), and the bacterial solution prepared with physiological saline so that the number of bacteria was 10'/ynl was injected into the tail vein of the mouse. 0.1 #//maunu) and infected. The efficacy was determined by whether the mice were alive or dead 7 days after infection.

Test results: FK-5 used in Test Examples 1 to 6 above
The 65-substance liposome formulations are each 8-substance.

Next, the method for producing the FK-'565 substance-containing liposome of the present invention will be explained with reference to Examples.

Example 1 L-σ-distearoylphonuphatidylcholine (60μ
Put the K'f8 dissolved solution in chloroform (4,74 t/mol) into a Nanu-type colben (100 ml capacity),
Chloroform is distilled off under reduced pressure in a water bath at 25 to 60°C to form a thin film on the inner wall of the Kolben.

On the other hand, F'-565 substance was dissolved in pH 7,4 phosphate buffered saline (4.3"i/ml), 0.
Filter using a 45μ membrane filter. This aqueous solution (4 t/) is placed in the above Kolben and shaken in a constant temperature shaker (60°C) until the thin film on the inner wall is peeled off.

Phosphate buffered saline is added to the resulting suspension, transferred to a polyethylene centrifuge test tube (2D*1 capacity), and centrifuged at 4°C, 1100, O'00X for 15 minutes. Remove the supernatant and transfer the precipitate to phosphate buffered saline (
20g/) and centrifugation were repeated twice. The resulting precipitate was dissolved in phosphate buffered saline (3.6 m
1) to obtain a liposome preparation of FK-565 substance. The concentration of FK-565 substance in this liposome preparation was 100μ9/ml, and the average particle size of the liposomes was 5μ.

The above phosphate buffered saline contains sodium chloride (80, [17F), potassium chloride (1,94y), dihydrogen phosphate dodecahydrate (22,92F), and potassium dihydrogen phosphate. (1,91P) was dissolved in distilled water to make a total amount of 101.

Example 2 L-α-dinutearoylphonuphatidylcholine (42μ
mo/L/) and phosphatidylserine (18 μmol)
Using a solution dissolved in chloroform (5.1 g+/) and an aqueous solution (4 ml) obtained by filtering a pH 7.4 phosphate buffered saline solution (600 μ9/ml) of FK-565 substance through a 0.45 μ membrane filter,
When treated in the same manner as in Example 1, a liposome preparation of FK-565 substance is obtained. FK-56 in this liposome formulation
The concentration of the five substances was 20μy/ml, and the average particle size of the liposomes was 25μ.

Example 3 Egg yolk lecithin (847+ moles) and phosphatidylserine (66 μmol) v) chloroform (11,9 m)
1) is treated in the same manner as in Example 1 to form a thin film on the inner wall of Kolben. On the other hand, FK-, -56
5 substances in pH 7.4 phosphate buffered saline solution (500
μ9/ml) through a 0.45μ membrane filter
The aqueous solution ('4 ml) filtered through the solution was placed in the above-mentioned Kolben, and stirred and shaken at room temperature until the thin film on the inner wall of the Kolben peeled off. Phosphate buffered saline (20 g/) was added to the resulting suspension, transferred to a polyethylene centrifuge tube ('50 vrl capacity), and incubated at 4°C, 100 ml.
, OD OXP for 115 minutes.

The supernatant was removed, the precipitate was redispersed in phosphate buffered saline (25 liters), and centrifugation was repeated twice. The resulting precipitate was dissolved in phosphate buffered saline (6.3g+/)
to obtain a liposome preparation of FK-565 substance. The concentration of FK-565 substance in this liposome preparation is 20p9/ml, and the average particle size of the liposome is 2.
It was 4μ.

Example 4 Egg yolk lecithin (26 μmol) and cholenutelo-tLy
(131tMo/L/ ) to Ji:X, Chi/L/ :r
--f) The solution dissolved in V (6 Ml) is placed in an eggplant-shaped colben (50 g+/vol.). In addition, FK-56
Phosphate buffered saline solution of 5 substances (400μy/tanl
, 2g+/) and sonicate for 5 minutes at 4°C.

Diethyl ether is distilled off from the resulting emulsion in a 25°C water bath under reduced pressure. The resulting suspension is filled into dialysis cellulose tubing and dialyzed in approximately 500 times the volume of phosphate buffered saline at 4°C for 48 hours. Furthermore, during this time,
The phosphate buffered saline is changed 5 times to increase dialysis efficiency. After the dialysis is completed, the liposome preparation of FK-565 substance is recovered from the tube. The concentration of FK-565 substance in this liposome preparation was 100 μy/ml, and the average particle size of the liposome was 2.6 μ.

Claims (1)

[Claims] A liposome preparation containing FK-565 substance represented by chemical structural formula: