JPH03173814A - Liposome preparation - Google Patents

Abstract

PURPOSE:To obtain a liposome preparation liable to be englobed with monocyte and macrophage and inducing the property of the sealed bioactive substance in improved efficiency by sealing a bioactive substance in a liposome containing an alkylmannoside. CONSTITUTION:Prescribed amounts of a liposome membrane component substance and an alkylmannoside (e.g. mannoside having 14-18C alkyl group) are solubilized with a proper organic solvent such as chloroform and the solvent is removed under reduced pressure. A solution containing a specific amount of a bioactive substance is added to the obtained lipid membrane to prepare a suspension of liposome. The amount of the alkylmannoside is preferably about 0.1-10 pts.wt. per 1 pt.wt. of the lipid. The bioactive substance is muramyl dipeptide, interferons, macrophage colony stimulation factor, granulocyte macrophage colony stimulation factor, etc., exhibiting activation action by the englobement with macrophage.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is a novel liposome preparation, more specifically, it is easily phagocytosed by monocytes and macrophages, and more effectively induces the properties of the encapsulated biologically active substance. Regarding liposome formulations.

Prior art and its challenges In the mid-1960s, when Bangham of the UK redispersed naturally derived phospholipids in water, a bilayer membrane with the same basic structure as the plasma membrane of cells was formed, and this Since discovering that the vesicles have the same closed vesicle morphology as the bulk structure, the vesicles have been called liposomes (somes) made of lipids.
As a membrane model or as a microcapsule, the fluidity and barrier capacity of the membrane (membrane permeation barrier) can be evaluated.
It is widely used as a research material for the structural characteristics of cell membranes, and for research on animal functions of cells such as aggregation and fusion, and active research is being conducted on it.

On the other hand, research on the process and mechanism of metastasis formation by cancer has been actively conducted in recent years, and the details of the cancer metastasis mechanism are being elucidated not only from the viewpoint of cancer cell-side factors but also from the perspective of host-side factors. Among these, the non-specific tumor effects of activated macrophages are attracting attention as a trend in cancer immunology. Activated macrophages can distinguish between normal cells and tumor cells, and therefore can selectively destroy tumor cells. Furthermore, it has been suggested that its cytotoxicity may overcome the drug resistance that often appears during cancer treatment or the heterogeneity of metastatic cancer. Furthermore, biologically active substances such as muramyldipentides (MDP) and colony-stimulating factors, especially M-C8F, have the ability to activate the macrophage system, and from the above point of view, these substances have anti-inflammatory properties. Clinical applications are expected as cancer drugs, immunostimulants, and other pharmaceuticals.

The physiological effects of the majority of biologically active substances or the above-mentioned monocyte-macrophage system activating substances, etc., exert their activation effects in vivo, while the biologically active substances such as these are directly activated. When administered into a living body, it is immediately degraded without exhibiting its effect, making it difficult to achieve a sufficient therapeutic effect.

In order to solve the above-mentioned problems, various methods have been investigated, such as the development of derivatives with long blood half-lives and administration forms, and the application of liposomes to the biologically active substances mentioned above has solved the problems. It is expected to be a powerful means to solve this problem.

Problems to be Solved by the Invention In order to solve the above problems, the present inventors have conducted extensive research and found that liposomes containing alkylmannosides contain biologically active substances, particularly MDP, M-C8F, etc. monocytes-
It has been discovered that encapsulating a macrophage system activating substance is effective in solving the above problems, and the present invention has now been completed.

Means for solving the problem, that is, the present invention relates to a liposome preparation characterized in that a biologically active substance is encapsulated in a liposome containing an alkylmannoside.

The liposome preparation of the present invention can exhibit excellent macrophage activation ability as described above.

The present invention also provides liposome preparations that can exhibit such excellent effects, liposomes useful as carriers for macrophage activating agents for the production of these preparations, and methods for producing them.

The liposome preparation of the present invention will be described in detail below. The preparation can be produced by encapsulating a biologically active substance in a liposome containing an alkylmannoside.

Examples of the alkylmannosides used here include mannosides having an alkyl group having 14 to 18 carbon atoms, typically an octadecyl group known as tetradecyl, pentadecyl, hexadecyl, hepta.

The liposome containing the alkylmannoside is not particularly limited as long as it contains the alkylmannoside. Various known methods, for example, typically the portex method (Vorlex method)
ing method or H7dration met
hod) (Bangham, A, D,, 5ta
ndish.

M, M., & Watkins, J., C. J., Mol.
, Biol,, 13. 238 (1965)). Examples of the liposome-forming lipids (membrane components) include phospholipids such as phosphatidylcholine (phosphatidylethanolamine, phosphatidylserine, sifbominiline, egg yolk lecithin, soybean lecithin, etc.). As cholesterol, sterols such as cholestanol, as charged substances dicetyl phosphate, phosphatidic acid,
A membrane component substance may be formed by adding stearylamine or the like and further an antioxidant such as α-tocopherol.

The ratio of the membrane component substances of the liposome and the ratio of these and acetylmannoside to be used are not limited in any way, but preferably about 0 to 2 parts by weight of sterols per 1 part by weight of lipid. , about 0 to 0.2 parts by weight of a charged substance,
Alkylmannoside 0. An amount of 1 to 10 parts by weight is suitable.

The liposome produced using the above phospholipid and alkylmannoside is not particularly limited, and may be a multilamellar liposome (MLV).
vesicle), multilamellar vesicle (SUV; smau
nilamella vesicle), unilamellar vesicle (LUV.

large unilamella vesicle
), but multilamellar liposomes (MLV) are usually preferred from the viewpoint of particle size, etc., and the liposomes usually have a particle size of 0. A size of 2 to 2 μm is preferred.

The biologically active substance encapsulated in the liposome obtained as described above is not particularly limited as long as it has an activating effect by phagocytosis of macrophages; for example, muramyl dipeptide (MDP), muramyl tripeptide, Peptide (
MTP) and their derivatives, α-interferon,
Examples include interferons (IFN) such as β-interferon and γ-interferon, colony activating factors such as macrophage-colony stimulating factor (M-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). The amount of such biologically activating substance to be used is appropriately determined depending on the type thereof, the type and type of the membrane component substance of the liposome, etc., and is not particularly limited. Approximately 0.05~50μ
g, about 100 units to 1 million units for M-C8F, GM
- Select from the range of approximately 1 unit to 100,000 units for C8F, approximately 10 units to 1 million units for α-tFN, approximately 100 units to 100,000 units for β-IFN, and approximately 100 units to 100,000 units for γ-IFN. It is appropriate that the

The production of liposomes encapsulating the above biologically active substances is as follows:
For example, a predetermined amount of a liposome membrane component substance and an alkylmannoside are solubilized in an appropriate organic solvent such as chloroform, the solvent is removed under reduced pressure to create a membrane lipid, and then a predetermined amount of the biological This can be carried out by adding a solution containing a therapeutically active substance to prepare a liposome suspension.

The liposome obtained by encapsulating the biologically active substance in this manner can be used as it is as the liposome preparation of the present invention.

Of course, operations such as sterilization can be performed according to conventional methods, if necessary.

EXAMPLES Examples will be given below to explain the present invention in more detail.

Example 1 ■i) Production of cetylmannoside-modified liposome Hydrogenated egg phosphatidylcholine (eggPC1 manufactured by Nippon Fine Chemical), cetylmannoside (C-Man).

A lipid mixture containing Dicetyl phosphate (DCP, Hani Chemical), Cholesterol (CH, Kanto Kagaku Kogyo type) in a molar ratio of 2:3:1:4 and a total lipid amount of 50 μmol. The fluorescent dye N-(lyaminorhodamine-β-sulfonyl)dioleoylphosphatidylethanolamine (Ayanti
After adding 3% (mol) of 5% calf serum (Gibco) and distilling off the solvent under reduced pressure to create a thin lipid film, RPM containing 5% calf serum (Gibco) was added.
11640 medium [Nissui Pharmaceutical Co., Ltd., hereinafter referred to as "5% CRP"
(called MtJ) 1111! was added to prepare liposomes by stirring.

Furthermore, by aging the prepared liposome suspension, the above-mentioned fluorescent dye not encapsulated in the liposomes was removed.

ii) Production of cetylmannoside-modified liposomeseg
g A chloroform solution of lipids prepared by mixing PCXC-Man, DCP, and CH with a molar ratio of 2+3:1:4 and a total lipid amount of 250 μmol was placed in an eggplant-shaped flask, and the solvent was removed under reduced pressure using an Evavolet 9-. leave,
A thin lipid film was created.

Add 5 yll of 0.1mM inulin-PBS solution to this.

[3H]-Inulin (Nrw England Nuc
lear (DuPant Co, , U, S,
Add 10ttll of MLV
Liposomes were prepared.

The particle size of the prepared MLV liposomes was made constant using a 0.8 μm filter (Nuclepore, Nomura Microscience Co., Ltd.). After that, 0.2
211 PBS once using am filter (Nuclepore, Nomura Microscience Co., Ltd.)
Dialysis was performed for 2 days with exchange twice a day.

■ Production of cetylmannoside-unmodified liposomes ■i above
), Cetylmannoside-unmodified liposomes were prepared in the same manner using egg PC, DPC, and CH at a molar ratio of 5:1:4, respectively.

Example 2 Measurement of oligophagic capacity ■ Isolation of monocytes and alveolar macrophages After separating the centrifuged leukocyte fraction from the venous blood of a healthy person into mononuclear cells using lymphoside separation medium (Organonteknica), El trine John method using John rotor (manufactured by Hitachi, 5RR6Y) [3,
5one el al,, I.

N, T, J, Cancer, vol.
38. p495-500 (1986)) to obtain monocytes.

Human alveolar macrophages were harvested using bronchoalveolar lavage (T,
Naka7ama et al. J, p, N, Th
orac, Dis,.

vol, 18. pH-19 (1984)).

■ Measurement of oligophagic capacity Monocytes or alveolar macrophages were plated on 8-well chamber slides at a ratio of 105 cells/well, and after cultured for 1 hour, fluorescently labeled liposomes (Example 1
) was added at 100 nmol/well and cultured for 12 hours.

After culturing, the cells were washed with PBS, fixed with 1% paraformaldehyde, washed, and embedded in glycerin.

The fluorescence intensity of each monocyte and lung cavity macrophage is PI(O
It was measured with TORC0UNTER (NF Corporation, photon counting device) (Clinical Examination 33 (6) 646, 1989).

The results are shown in Table 1 below as relative values taking the poor eating rate of the control as 100%.

Table 1 From the above table, it can be seen that the cetylmannoside-modified liposome obtained in Example 1i) has a property that it is more easily phagocytosed by both monocytes and macrophages than unmodified liposome. .

Example 3 i) Production of MDP-encapsulated cetylmannoside-modified liposome egg-PC: C-Man: DCP: CH=2
A chloroform solution of lipids mixed with a composition of: 3: 1: 4 (molar ratio) and a total lipid amount of 50 μmol was distilled off under reduced pressure to form a thin lipid film, and then norMDP ( NorMDP-encapsulated cetyl mannoside-modified liposomes were prepared by the portex method by adding 1 xi of 5% CRPMI containing 20 t1 g of demethylated muramyl dipeptide (Ciba Geigy). Unencapsulated norMDP was removed by centrifugation.

ii) Production of MDP-encapsulated unmodified liposomes egg PC
:DCP:CH=5:1:4 (molar ratio) In the same manner as in i) above, a cetylmannoside-unmodified liposome encapsulating nαrMDP was prepared.

1ii) Measurement of tumoricidal cell activity The monocytes obtained in Example 2 were placed on a microtest plate (
(manufactured by Falcon) at a rate of 105 monocytes/well, and after culturing for 1 hour, a molar year of monocytes was obtained.

The purity after making the mori year was 99% or more due to the washing operation.

Furthermore, norMDP alone was added to the monocyte molar year, i) above.
Various amounts of the norMDP-encapsulated cetylmannoside-containing liposome obtained in step ii) and the norMDP-encapsulated cetylmannoside-unmodified liposome obtained in step ii) were added in various amounts as shown in Figure 1, and cultured for 24 hours under 5% CO2 conditions.

After culturing, wash and collect 104 monocytes/well (monocytes (E
) to tumor cells (T): E/T = 25 10/1) I-IUdR-labeled human A375 melanoma cells were added and cultured at 37°C for 72 hours.

After the completion of the above culture, the tumor cells were washed and the radioactivity of the tumor cells living at the bottom of the well was measured, and the cytotoxicity rate (%
) was calculated.

Control group radioactivity (cpm) The results are shown in FIG. Figure 1 shows the cell damage rate (%) on the vertical axis.
, the norMDP concentration (μg/yIl) is taken on the horizontal axis,
This is a diagram examining the influence of each active substance sample on the cell-killing effect of A375 cells by monocytes. ) shows the comparative sample, that is, the norMDP-encapsulated unmodified liposome obtained in the same ji), and (3) shows free norMDP, respectively.

From the above figure, it can be seen that the cell killing activity of monocytes against A375 cells is significantly enhanced by activation with the liposome sample of the present invention. In contrast, the monocyte activation ability of the comparative liposome sample was weak, and free norMD
It can be seen that in P, a large amount of addition of 0.1 μg/xi or more is necessary for monocyte activation.

Example 4 i) Production of M-C8F-encapsulated cetylmannoside-modified liposome In Example 3, CHO was used instead of 20 μg of norMDP.
M-CSF produced from cells (Unexamined Japanese Patent Publication No. 124663
M-CSF in the same manner using 10,000 units (No. Publication)
Encapsulated cetylmannoside-modified liposomes were prepared.

ii) Production of unmodified liposome encapsulating M-C8F M-C8
M-CSF-encapsulated cetylmannoside-unmodified liposomes were prepared in the same manner as in Example 511) using F100OO units.

1ii) Measurement of monocyte survival rate The monocytes obtained in Example 2 (1) were plated on a microtest plate (manufactured by Falcon) at a rate of 1 x 105 cells/well, cultured for 1 hour, and the monocytes obtained in Example 2 Got a ball mole layer.

Furthermore, free M-CSF, the present invention M-CSF obtained in the above i)
Encapsulated cetylmannoside-modified liposome sample and ii)
Each of the comparative M-C8F-encapsulated unmodified liposome samples obtained in 1 was added over time, and the survival rate of monocytes was assayed. This survival rate was calculated using the Mosmann method (T, Mosmann, J
our own of 1mmunologica1Me
thods, 15.55-63 (1983)].

The results after the first week of culture are shown in Figure 2. The reading plot shows survival rate (%) on the vertical axis and M-CSF concentration (unit/xll) on the horizontal axis.
), and the effect of maintaining monocyte survival rate by each sample was examined. (1) shows the cetylmannoside-modified liposome sample encapsulating the M-CSF of the present invention, and (2) shows the comparative non-modified liposome sample encapsulating M-CSF. , (3) indicate free M-CSF, respectively.

From the diagram reading, the liposome sample of the present invention is compared to the comparative sample and free M
- It can be seen that the survival rate of monocytes can be significantly increased compared to CSF.

Example 5 Testing of tissue distribution by administering cetylmannoside-modified liposomes Male Wistar rats (body weight 200±20 g) were used as experimental animals for testing tissue distribution. The rat was anesthetized with ether, cannulae were inserted into the femoral artery, vein, and bladder, and after surgery, it was fixed in a Pohlmann cage.

After awakening from anesthesia, labeled liposomes (modified liposomes obtained from Example 1 (ii) and unmodified liposomes obtained from Example 1) were administered at 0.5z to 200 g of rats.
11 was administered, blood and urine were collected over time, and the tissue distribution of the liposomes was examined 2 hours later.

The sites tested were blood, liver, lungs, lungs, kidneys, and urine.

First, 0.2xl of blood was collected, and after decolorizing with H2020,511, 0.5z of 2N KOH-isopropanol solution was added.
For urine, add purified water to make a total volume of 1011, then 1
．． Measure out 0xll and get H2020, 2ytl.

2N KOH-isopropanol solution 0. '2tl!
was added and left overnight at room temperature. then 10 on the blood sample
% acetic acid solution 17A', and also Ex-H (scintillation cocktail, scintisol Ex-H, Dojindo Laboratories) 1
0y/ was added.

After homogenizing the liver, add purified water and bring the total volume up to 50%
yl, take 1 xll, H2O20, 2xf
2N KOH-isopropanol solution 0.5zl,
Furthermore, Ex-H10z/ was added.

For lungs and kidneys, H2O20,1,xll, 2N KOH-
Isopropanol solution 2111 pancreas H2O22, v/
, 2N KOH-isopropanol solution 27A' was added and incubated overnight at 37°C.

Then, 10% each for lung, kidney, and lung samples.
of acetic acid and further added purified water to bring the total amount to 1. Oxl
After fractionating the LOyl, Ex-H10y/ was added.

After stirring each sample, radioactivity was measured using a liquid scintillation counter (LSC-602 manufactured by Aloka).

Table 2 below shows the results of examining the influence of lipid composition on the tissue distribution of liposomes 2 hours after intravenously administering liposomes encapsulating [3H]-inulin to rats.

Table 2 From the above table, it can be seen that approximately twice as much of the cetylmannoside-containing liposome is distributed in the liver. This is presumed to be because the liposomes are taken up by Kuppa cells, which are a type of macrophage present in the liver.

[Brief Description of the Drawings] Figure 1 is a diagram showing the effect of each sample on the cell-killing effect of monocytes on A375 cells according to Example 3, and Figure 2 is a diagram showing the effect of each sample on monocyte survival according to Example 4. This is a diagram examining the rate maintenance effect. (Above) Survival rate (olo) Spicy Moon Miko B Blue 9.5 (%)

Claims (3)

[Claims] (1) A liposome preparation comprising a biologically active substance encapsulated in a liposome containing an alkylmannoside. (2) The liposome preparation according to claim (1), wherein the biologically active substance is muramyl dipeptide. (3) The liposome preparation according to claim (1), wherein the biologically active substance is M-CSF.