JPS6314717A - Liposome preparation - Google Patents

Abstract

PURPOSE:A liposome preparation capable of collecting a chemotherapeutic agent at the affected part by carrying out magnetic field control from the outside and making the chemotherapeutic agent act specifically and concentrically, containing the chemotherapeutic agent and magnetic iron oxide in particles and/or membranes. CONSTITUTION:0.001-0.7pt.wt. based on 1pt.wt. complex phospholipid forming liposome of a chemotherapeutic agent (e.g. carcinostatic agent, enzyme, enzyme inhibitor, anti-inflammatory drug or immunosuppressor) and 0.001-0.7pt.wt. magnetic iron oxide (Fe3O4) are contained in particles and/or membrane of liposome preparation. Magnetosome obtained by cultivating a magneto-taxis microorganism and separating the mold or magnetic iron oxide obtained by further purifying the magnetosome is preferably used as the magnetic oxide.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a new form of liposome preparation, and more particularly, to a liposome containing magnetic iron oxide and a chemotherapeutic agent in the particles and/or membrane. Regarding the preparation, such a preparation is effective in selectively transporting the chemotherapeutic agent to the affected area by controlling the magnetic field from the outside, and maintaining a high concentration of the chemotherapeutic agent locally.

[Prior art]

Generally, chemotherapeutic agents used for various diseases do not have selectivity between affected areas and normal areas, and the occurrence of severe side effects is unavoidable.

Additionally, targeting therapy using monoclonal antibodies has been devised to solve this problem. (Unexamined Japanese Patent Publication No. 5
8-134032).

However, monoclonal antibodies can only be used against the same type of antigen, and different monoclonal antibodies must be prepared for each antigen, and the production of monoclonal antibodies is very laborious and time-consuming. Furthermore, it is considered to be extremely difficult to produce monoclonal antibodies using human-human hybridomas, which are highly safe for humans, using current technology.

[Problem that the invention seeks to solve]

In view of the above-mentioned circumstances, the present inventors have conducted extensive research to selectively exert the chemotherapeutic agent on the affected area, and have found that magnetic iron oxide and the chemotherapeutic agent are contained within the particles and/or in the film. The present invention has been completed based on the discovery that a liposome formulation achieves the above object.

[Means for solving problems]

That is, the present invention is a liposome preparation containing magnetic iron oxide and a chemotherapeutic agent within the particles and/or the membrane.

Such preparations can selectively transport the chemotherapeutic agent to the affected area by controlling the magnetic field from the outside, and can maintain a high local concentration of the chemotherapeutic agent.

Examples of chemotherapeutic agents in the present invention include anticancer agents such as cyclophosphami I, busulfan, actinomycin D1, daunomycin, adriamycin, mitomycin C1, bleomycin, 7-ocartinostatin, and vincristine; enzymes such as urease, asparakinase, catalase, and amylomycin; Glucosidase, carbonisoquanchi Ilase, neuraminidase, etc. Coenzyme A1 vitamin B12, ubiquinone, biotin, etc. Enzyme inhibitor clopeptin, thiolstatin D1 pebuscutin 010 ipebutin, chymosfutin, leupeptin, nodeoxylicirimycin, etc. As an anti-inflammatory steroid, dexamethacin phosphate, dexamethacin acetate, cortisone, etc.
Examples of non-steroidal anti-inflammatory agents include mefenamic acid, indomethacin, merilluxe I, etc., and immunosuppressants include glycocorticoids, 6-mercaptopurine, imran, cyclophosphamide 1, adriamycin, etc.

Liposomes are endoplasmic reticulum formed by a lamellar phase composed of complex lipids, and can contain (embed) both water-soluble and fat-soluble substances.

That is, the water-soluble substance is contained in the aqueous phase within the liposome, and the fat-soluble substance is contained in the lamellar phase forming the liposome. In addition, these substances may be chemically and physically adsorbed on the surface of the lamellar phase, and in the present invention, the above three cases are collectively referred to as "containment-1."

As a method for preparing liposomes, the port swing method (A,
D., Bangham, J., Mo1. Biol,,13,
238 (1965)), sonication method (C, I
luang, Biochem, , 8.344 (+
969)), Prehesicle method (Il, Trauble,
Neurosci, Res, Prog, Bull,, 9
, 273. (1971) ), ethanol injection method (
S, Batzri, Biochem, Biophys
, Acta+ 298+ 1015 (1973))
, French press extrusion method (Y, Barenholz, F
EBS Lett, 99, 210 (1979)
), cholic acid removal method (T, Kagawa, J, Bio
Chem, 246.5477 (1971))
, Triton X-100 hatch method (W, J, Gerri
tsen, F! ur.

J. Bjochem, 85, 255 (1978)
), Ca2 fusion method (O9Papagadjopou
los, Biochem, Biophys, octa,
394, 483 (1975)), ether injection method (
D, Deamer, Biochem, Bio
phys, ^eta, 443, 629 (1976)
), annealing method (R, Lawaczeck, B
iochem, old ophys, Acta, 443,31
3 (1976>), freeze-thaw fusion method (M, Kas
ahara, J., Biol, chem,, 252.73
84 (1977)), W/O/W emulsion method (S, Matsumoto, J, Goli
oid,InterfaceSci,,62,149
(1977) ), reversed-phase evaporation method (F, 5zoka.

Proc, Natl, 8cad, Sci', ll5A,
Although many methods are known, such as 75.4+94 (197B), any preparation method may be used to contain the magnetic iron oxide and chemotherapeutic agent according to the present invention, and is not limited to these methods. .

Examples of the complex lipids used in the present invention include lecithin, cephalin, phosphatidylcholine, sphingomyelin,
Natural phospholipids such as plasmalogen, cimilistoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, dicetylphosphe-1, synthetic phospholipids such as stearylamine, digalactosyl diglyceride, 6-sulfoquitubosyl diglyceride, galactosylceramide, glucosylceramide, etc. natural glycolipids, synthetic glycolipids such as glucosyl dipalmitoyl glycerol, cellubiosyl dipalmitoyl glycerol, maltosyl civalmi Fyl sugars, or hydrogenated lecithin sugars in which the unsaturated carbon chains of naturally derived lecithin are saturated with hydrogen. Can be mentioned. One or more of these may be selected and used.

In order to enhance the dispersion stability of liposomes, it is desirable to impart an electric charge to the complex lipid lamellar phase. In this case, when imparting a negative charge, a negatively charged lipid such as phosphatidyl heptaphosphorus or dicetyl phosphate 1 may be used, and when imparting a positive charge, a positively charged lipid such as stearylamine may be used.

In order to stabilize the lysopome formulation of the present invention, a sterol can be further added. Such sterols include, for example, cholesterol, β-sitosterol, stigmasterol, campesterol or mixtures of sterols extracted from plant materials.

There are no particular limitations on the amount of the components contained in the liposome preparation according to the present invention, both magnetic iron oxide and chemotherapeutic agent, but preferably 1 complex phospholipid that can form liposomes.
On the other hand, both have a compounding ratio of o and oot to 0.7 (weight ratio), respectively. If the blending ratio exceeds this range, the components that cannot be contained will be present in the dispersion medium.

Further, the shape and size of the magnetic iron oxide are not particularly limited, but preferably 100 to 0.1 μm. If the size exceeds this, the stability of the liposome preparation itself according to the present invention will deteriorate, and if it becomes smaller than 100 people, the response to the magnetic field will become weaker, and the magnetic force of the liposome preparation will also become weaker, which is not preferable. .

As such magnetic iron oxide, the following are particularly preferable. That is, what is generally called a magnetotactic microorganism, such as Sprillum volutans ATCC1
After culturing 9554 according to a conventional method, a magnetosome is separated from the bacterial cells. This product may be further prepared into II according to a conventional method and used. The magnetotactic microorganism to be used is not particularly limited, and any microorganism having magnetorems in its cells, such as bacilli, cocci, and spiral bacteria, may be used. Of course, Magnetreme may be contained in the liposome preparation according to the present invention as it is.

The liposome preparation of the present invention can also contain ordinary pharmaceutical and cosmetic ingredients as long as they do not destroy the form of the liposome.

〔Effect of the invention〕

The liposome preparation of the present invention containing a chemotherapeutic agent and magnetic iron oxide can be concentrated at the affected area by controlling the magnetic field from the outside, making it possible for the chemotherapeutic agent to act specifically and intensively. be.

〔Example〕

Example 1 Magnetic iron oxide was obtained as follows. That is, Spir
illum volutans ATTC19554, 3O7! The cells were cultured in a jar fermenter using a medium having the following composition.

MgSO4・71f20. 20 gMnSO
4・N20 40 gPeCI3・6 N2
0 180 mg(NH4) 2 SO42
0g Succinic acid 33.6g
Peptone 100g water
20 β culture is
The cells were cultured for 5 days at a culture temperature of 30° C. while blowing N2 gas containing 5% 02 gas. Bacterial cells were collected from the obtained culture solution, reacted with 0.2% lysozyme solution at 37°C for 1 hour to dissolve the cell wall, and further incubated with 5 M NaOH for 12 hours.
After time treatment, magnetic particles were isolated.

Next, in a 501p eggplant flask, 0.1 g of dipalmitoyl lecithin and 4 μg of cetyl phosphate were dissolved in 3 me of chloroform, and then 500 μg of actinomycin D (hereinafter referred to as AD) was added and the chloroform was distilled off using a rotary evaporator. A composite lipid film was obtained on the bottom wall of the flask. This was dried in a vacuum desiccator for 2 hours to completely distill off chloroform. To this was added 10 mQ of a 0.1 wt% aqueous solution of magnetic iron oxide particles, and
After stirring for a minute, liposomes were formed by vigorously shaking using a portex mixer to obtain a liposome dispersion.

Liposomes were then isolated using a concentration gradient (0-20%) of dextran to remove uncontained actinomycin D and magnetic iron oxide particles. This was diluted appropriately to obtain a liposome preparation (Fe-LiP-AD).

[Test Example 1] C3H/lle mouse (male, 4 weeks old) 5x10 injected into the peritoneal cavity
``Ehrlich carcinoma tumor cells were implanted. After 24 hours, 5
0PI! After intravenous administration, a small magnet was attached to the abdomen of the mouse with tape so that the liposomes according to the present invention would be collected around the abdominal cavity of the mouse, and the anticancer effect was investigated.

For comparison, AD as is, liposomes containing only magnetic iron oxide (Fe-Lip), and liposomes containing only AD (Lip-AD) were administered intravenously to examine their effects on cancer. Before attaching the small magnet to the mouse, the team checked which side would attract the magnetic iron oxide particles. Table 1 shows the results of examining the therapeutic effect of the above formulation on tumors.

Note that each liposome for comparison was prepared according to Example 1.

As is clear from the table, the liposome formulation according to the present invention has excellent therapeutic effects.

(Margin below) *All cured mice survived for more than 100 days.

(The following is a blank space) Example 2 Magnetic oxidation obtained in the same manner as in Example 1 after dissolving 70 ■ of egg yolk lecithin, 3 O ■ of cholesterol, and 4 ■ of dicetyl phosphate in diethyl ether 3 in a 50-Eggplant flask. A W10 emulsion was obtained by adding a 5% dexamethacin phosphate solution 1- in physiological saline containing 0.1% by weight of iron particles and then irradiating it with ultrasonic waves. The diethyl ether was distilled off using a rotary evaporator to form a gel, and after shaking lightly, the remaining diethyl ether was completely distilled off to obtain a thick liposome dispersion. Dexamethacin phosphate not contained in the liposomes was removed using a Sepharose 4B column according to a conventional method, and then diluted appropriately to obtain a liposome preparation.

[Test Example 2] The anti-inflammatory activity of the liposome preparation obtained in Example 2 was evaluated. That is, a physiological saline solution of Example 2 liposome preparation and dexamethasone phosphate I was intravenously administered to Wistar rats (male, 5 rats per group, weight 180 g) at a dose of 3 kg/kg, and administered to the hind limbs. A small magnet was attached to the bottom of the foot. After 18 hours, carrageenan edema was induced by administering a 1% carrageenan saline solution [0,]me@under the hind limbs. After administration, the footprint volume was measured after a certain period of time, and the rate of increase in footprint volume (
V,) was calculated. As a control group, the rate of increase in footprint volume (Vo) was measured when carrageenan was similarly administered to rats to which only physiological saline was intravenously administered, and (Vo V+)
/ Vo The larger this value is, the higher the anti-inflammatory activity is. Table 2 shows the measured values 5 hours after carrageenin administration.

Table 2 As is clear from Table 2, the liposome preparation according to the present invention has excellent therapeutic effects.

Claims (3)

[Claims] (1) A liposome preparation containing a chemotherapeutic agent and magnetic iron oxide (Fe_3O_4) within particles and/or membranes. (2) Claim 1 in which the chemotherapeutic agent is an anticancer agent, an enzyme, a coenzyme, an enzyme inhibitor, an anti-inflammatory agent, or an immunosuppressant.
Liposomal formulations described in Section 1. (3) The liposome preparation according to claim 1, wherein the magnetic iron oxide is a magnetic iron oxide isolated and purified from a magnetotactic microorganism or a magnetosome isolated from a magnetotactic microorganism.