JPH04159216A - Liposome containing glucosamine derivative as film material - Google Patents

Abstract

PURPOSE:To obtain a cationic liposome having excellent retention in blood, a long half-life of drug in blood and low toxicity, containing a specific glucosamine derivative as a film material. CONSTITUTION:A liposome which contains a glucosamine derivative shown by the formula [R<1> and R<2> are H or CO(CH2)nCH3 (n is 10-22 integer); R<3> is H or lower alkyl; m is 0-3 integer; with the proviso that R<1> and R<2> are not H at the same time] or a salt thereof, preferably 6-O-palmitoyl-D-glucosamine methylglucoside as a film material, retains preferably a polypeptide having superoxide dismutase (extremely oxidative disproportionating enzyme) activity as a drug to be sealed in the liposome, has much lower toxicity than that of conventional cationic liposome, high anionic or neutral sealing ratio, a long half-life in blood and little transition to organs.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is based on the following formula (1) (wherein R1 and R2 are hydrogen atoms or -CO(CH,),
tcH1 group (n means an integer of 10 to 22), R3 means a hydrogen atom or a lower alkyl group, and m means an integer of 0 to 3. However, R1 and R2 are not hydrogen atoms at the same time.) The present invention relates to a liposome containing a glucosamine derivative or a salt thereof as a membrane material.

[Conventional technology]

Liposomes are hollow, closed vesicles consisting of a lipid bilayer membrane produced by dispersing a polar lipid thin film formed from phospholipids etc. into an aqueous solution, and are basically similar to the lipid bilayer membrane structure of biological membranes. Since they have the same structure, they are regarded as a type of microcapsule that is biocompatible, and various drugs and enzymes are encapsulated within the capsules.
Attempts have been made to administer it to living organisms.

On the other hand, various substances are known as drugs to be encapsulated in liposomes depending on the purpose, and an example thereof is superoxide dismutase (superoxidative dismutase; 5OD). . Superoxide anion (02-) is a type of active oxygen produced in the body during the reduction process of molecular oxygen, and is produced in response to external stimuli such as antigen-antibody complexes.
By oxidizing and decomposing foreign substances, it is useful for sterilization and detoxification. However, when active oxygen is produced in excess, it acts as a powerful harmful and inflammatory factor, causing inflammatory diseases such as rheumatism, Bartchet's disease, and Crohn's disease.
It is one of the causes of diabetes or cancer disease (Drug Delivery System;
System, 2(1), 1987), S
OD is an enzyme that inactivates these active oxygen species.

However, SOD has an extremely short half-life in the blood (
(about 6 minutes) and lacks stability.

Therefore, in order to improve these drawbacks, various attempts have been made to stabilize and release SOD by encapsulating it in liposomes (for example, JP-A-63-211222, JP-A-1-175944).

1-238537, 1-246225).

On the other hand, phospholipids such as phosphatidylcholine have traditionally been used as membrane materials to form liposomes, but recently, these have been used to improve drug encapsulation and cell adhesion of liposomes. Attempts have also been made to impart a positive charge to the liposome membrane surface by adding charged substances such as stearylamine to phospholipids (Japanese Unexamined Patent Publication No. 63-7
No. 7824, JP-A-1-106829, “Drug Delivery System J, 12(1), 4l
It has also been suggested that these cationic liposomes have better retention in blood than neutral or anionic liposomes (Eur, J, Bi
ochem, 47, 179-185 (1974))
.

[Problem to be solved by the invention]

However, stearylamine, which is commonly used in the production of these cationic liposomes, has a serious drawback in that when applied to biological systems, side effects such as convulsions, that is, toxicity are expressed.
, 31, 173-179 (1977)), but it was not practical.

The present inventors previously published It (Patent Application Hei 1-2).
Further research on the new glucosamine derivative (No. 8933) revealed that cationic liposomes prepared using this derivative have excellent blood retention, extend the half-life of the drug in the blood, and have a low surface resistance. This discovery led to the completion of the present invention.

[Means to solve the problem]

The present invention is based on the following formula (1) (wherein R1 and R2 are hydrogen atoms or -CO(CH2),
It means a CH3 group (n means an integer of 10 to 22), and R3 means a hydrogen atom or a lower alkyl group.

m means an integer from 0 to 3. However, R' and R2 are not hydrogen atoms at the same time.) The present invention relates to a solusome containing a glucosamine derivative or a salt thereof as a membrane material.

Here, the lower alkyl group includes a methyl group, ethyl group, propyl group, isopropyl group, butyl group, and the like.

It is an alkyl group having 1 to 4 carbon atoms, and particularly preferably a methyl group. Compound [1] is also free of Iai!, such as fA acid, sulfuric acid, phosphoric acid, etc. ! Alternatively, pharmacologically acceptable salts with a high sI content such as tartaric acid, maleic acid, fumaric acid, and succinic acid can be produced.

The compound represented by formula (I) above is basically composed of a hydrophobic part (RI and R2) made of higher fatty acid residues and a hydrophilic part made of glucosamine residue, and the hydrophobic part As long as the higher HMB acid residue constituting the carbon chain has a sufficient carbon chain to impart hydrophobicity, the purpose can be fully achieved. That is, the higher fatty acid residues R1 and R2 are groups for imparting hydrophobicity to the molecule, and like the phospholipid that constitutes the majority of liposomes, the fatty acid residue of compound [1] has 10 or more carbon atoms, Preferably 12 to 24, i.e. n=10
-22 is desirable. In addition, the purpose of the glucosamine moiety is to impart hydrophilicity, and it may be in any form as long as it does not interfere with the ability to form liposomes.
However, monomers (m=o), 22-mers (rn
= 1), trimer (m = 2), or 4] 1 body (m =
3), particularly preferred are monomers in which m is 0 or 1, and 2-mer monomers.

Representative examples of the compound represented by the general formula (1) are as follows, but the invention is not limited thereto.

6-0-Lauroyl-D-glucosamine methyl glycoside 6-0-Myristoyl-D-glucosamine methyl glycoside 6-0-Valmitoyl-D-glucosamine methyl glycoside 6-0-stearoyl-D-glucosamine methyl glycoside 3.6-di0 -Lauroyl-〇-Glucosamine Methyl Glycoside゛3.6-Di-O-Myristoyl-〇-Glucosamine Methyl Glucoside 3.6-D-O-Valmitoyl-D-Glucosamine Methyl Glycoside 3.6-Di-O-Stearoyl-D- Glucosamine methyl glycoside 6.6'-di0-valmitoyl-〇-gelcosamin (1→4)-β-D-glucosamine methyl glycoside 3-0-valmitoyl-〇-glucosamine methyl glycoside 6-0-valmitoyl-〇-glucosamine liposome can be broadly classified into liposomes (multilamellar vesicles,
MLV, multilamellar vesic
le), small unilamellar liposomes (small unilamellar vesicles, SUV, small unilam
llarvesicle), large unilamellar liposomes (large unilamellar vesicles, LUV, large unil
amellar vesicle), etc., but the liposome of the present invention may have any structure among these.

The lipid for mIIing the liposome may be any of the lipids commonly used for forming liposomes, such as phosphatidylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylethanolamine. Examples include natural or synthetic phospholipids such as fatidylserine, phosphatidylinositol, sphingomyelin, egg yolk lecithin, and soybean lecithin, hydrogenated Ws-added phospholipids, and glyceroglycolipids. These may be used alone or in a mixture of two or more.

Furthermore, in order to stabilize the membrane structure, a sterol stabilizer such as cholesterol or an antioxidant such as tocopherol or β-carotene may be appropriately selected and used.

The glucosamine derivative or its salt represented by the general formula (13) used in the present invention is a new substance, and we have previously published W (
An example of the manufacturing method is as follows.

Known amino sugar or its salt! II! is reacted with benzyloxycarbonyl chloride in a solvent such as pyridine to preserve the amino group at the 2-position, and then reacted in methanol using TsOH or the like as a catalyst to methoxylate the 1-position. Furthermore, by reacting with higher fatty acid chloride in a solvent such as pyridine, 6
It can be produced by esterifying the position and/or the 3-position and then removing the amino protecting group at the 2-position.

Although the composition ratio of the membrane material in the liposome according to the present invention is not particularly limited, for example, the glucosamine derivative represented by the general formula (1) is 0.0% to 100 parts by weight of phospholipid. The amount is 5 to 30 parts by weight, preferably 1 to 25 parts by weight. Glucosamine derivative is 0. If the amount is less than 5 parts by weight, the effect as a cationic liposome will not be sufficient, and if it is added in excess of 30 parts by weight, it is unreasonable in terms of cost.
On the contrary, the ability to form liposomes may be reduced.

In addition, 0 to 30 parts by weight of a stabilizer such as cholesterol,
If necessary, 0 to 5 parts by weight of other compounding agents such as antioxidants may be added.

The liposome of the present invention can be prepared according to known methods commonly practiced in the art.

For example, membrane materials such as phospholipids, glucosamine derivatives, other membrane stabilizers as necessary, and charged substances.

A bleaching inhibitor and the like are dissolved in an organic solvent and placed in a flask, and the solvent is distilled off under reduced pressure to form a lipid thin film on the inner surface of the flask, followed by vacuum aIL drying.

To this, an rIfIJ solution containing a drug in a buffer such as Tris-HCl is added to wet the thin film, shaken, and preferably subjected to ultrasonication to form a dispersion, and further remove the remaining residue. After the encapsulated drug and the like are removed by centrifugation, gel filtration, or ultra-transient operation, the drug is redispersed in an isotonic aS solution or the like to obtain a drug-encapsulated liposome dispersion.

This drug-loaded liposome dispersion can be used as it is as an injection, but if necessary, it can be lyophilized to prepare a Kawasaki-type injection. If it is desired to adjust the particle size of liposomes, any known method such as pressure transfer method (extruder method), gel transfer method, centrifugation method, etc. may be selected and used as appropriate.

The particle size of the liposomes used in the present invention is not particularly limited, but is preferably 0.3 μm or less, more preferably 0.3 μm or less, and more preferably 0.3 μm or less. It is preferably 2 μm or less.

Furthermore, regarding the drug held in the liposome of the present invention, SOD was previously described as an example.

The drug is not limited thereto, and various water-soluble or fat-soluble drugs can be used. However, since the liposome itself has a positive charge, it is preferable to use neutral or anionic drugs in addition to the above-mentioned SOD, anti-inflammatory drugs such as indomethacin, and oxygen transport agents such as hemoglobin. , enzymes such as urokinase, antibiotics such as doxycycline hydrochloride and gentamicin sulfate, insulin,
Examples include hormonal agents such as 80TH (adrenocorticotropic hormone) and calcitonin, and anticancer agents that take advantage of their long retention time in the blood.

(The following is a blank space) [Reference Examples and Examples] The present invention will be explained in more detail by referring to Reference Examples and Examples below.

Reference Example 1 Method for producing 6-0-valmitoyl-D-glucosamine methyl glycoside N-benzyloxycarbonyl-D-glucosamine methyl glycoside was obtained from D-glucosamine according to a known method, and the N-benzyloxycarbonyl was dissolved in pyridine (50 ml). -D-glucosamine methyl glycoside (9
, 3 g) and valmitoyl chloride (8 ml) are added and stirred at room temperature for 24 hours under a nitrogen gas atmosphere. After the reaction is complete, the reaction mixture is poured into 10% water-cooled hydrochloric acid and extracted with ethyl acetate. Thereafter, the extract was washed with saturated sodium bicarbonate (N a HCO3), brine (salt solution), dried over anhydrous sodium sulfate (N a2 SO,), and the solvent was removed to obtain the crude product. , further recrystallized from ethyl #acid solution to obtain N-benzyloxycarbonyl-
6-〇-valmitoyl-D-glucosaminomethyl glycoside (7.65 g) was obtained. This N-benzyloxycarbonyl-6-〇-valmitoyl-D-glucosamine methyl glycoside (1.35 g) was mixed with methanol (50 g).
m'l), add a catalytic amount of 5% Pd-C, perform catalytic reduction at room temperature and normal pressure for 24 hours, and after the reaction is finished, pass through this to remove the solvent, and the residue is purified by silica gel column chromatography. The target compound 6-0-balmitoyl-D-glucosamine methyl glycoside (874
mg) was obtained. Take rate 85%.

m, p, 7 70. 5-71.5℃ Elemental analysis: C11N Actual value 64.24 Gourd 10.84% 3.04% Calculated value -64,00% 10.51 made 3.25% (' C
Calculation as 23Ha 30 e N M) I R (K
B r ): 3350cm-',
2920c+a-', 2850ci+-', 17
30cm-', 1460cm-'M S (FA
B): 432 (M+1)'''IH-NMR (DM
SO-d6) δ (p p m): 5.15 (a, III)
;5.00(s+,III);4.51(d+J=3.
411z, 111); 4.30 (d, J=10.61
1z, III): 4.04 (dd, J=6.611
z, J=6.811z, l1l): 3.53(m,
III): 3.26 (s, 3H); 3.10 (■, 2
It): 2.40 (m, 111): 2.29 (t, J=
7.211z, 2H); 1.51 (m, 2H); l,
24(b, 248); 0.86(t, J=6.0IIz
, 311).

Reference Example 2 Process for producing 6-0-lauroyl-〇-glucosamine methyl glycoside According to Reference Example 1, the title compound was obtained using lauroyl chloride instead of balmitoyl chloride.

Elemental analysis: CHN Real 93 @ 60.34% 9.95% 3.63 Calculated value" 60.80% 9.87% 3.73% ('
Calculated value as C++*HsvOeN) Reference Example 3 Process for producing 6-0-myristoyl-D-glucosamine methyl glycoside Following Reference Example 1, the title compound was obtained by using myristoyl chloride in place of balmitoyl chloride.

mn p, : 65-70℃ Elemental analysis: CH
N Actual FJ (+1 62.18% 10.27% 3.33
% Calculated Value" 62.53% 10.17% 3.47 Gourd (' Calculation as C21Ha, Os N (11) Reference Example 4 Production method of 6-0-stearoyl-D-glucosamine methyl glycoside According to Reference Example 1 The title compound was obtained by using stearoyl chloride in place of valmitoyl chloride.

MS: 459.3542 (M") (C2sH
A, OaN L/r) Total
Preparation of (1→4)-β-D-glucosamine methyl glycoside D-gelcosamino-(1→4)-β-D-glucosamine hydrochloride (2.5 g) and sodium hydrogen carbonate (2.1 g)
Mixed solution #: (water: methanol = 3: 4 V/V
, 70 ml) and then benzyloxycarbonyl chloride (PhCH20COC1) (2.4 g; 2
m1) was added and stirred at room temperature for 2 hours. Thereafter, the product was passed through, washed twice each with purified water and dichloromethane, dried under vacuum, and then recrystallized with methanol. As a result, N,
N'-dibenzyloxycarbonyl-D-gelcosamino-(1→4)-β-D-glucosamine (62 mg) was obtained.

This compound (1.5 g) was dissolved in pyridine (15 ml) and mixed with #acid anhydride (10 ml H approx. 0.11 mol
) was added and stirred at room temperature for 12 hours. After the completion of the reaction, 10%
Hydrochloric acid was added, and the mixture was extracted with ethyl acetate. Wash the extract with saturated saline and dilute with anhydrous 1lIle! After drying with sodium, the solvent was removed and N,N#-dibenzyloxycarbonyl3. 4. 6. 1', 3', 6'-
Hexa-0-acetyl-D-gelcosamino-(1→4)
-β-D-glucosamine (1°88 g) was obtained. Furthermore, this compound (1.5 g) was added to dry dimethyl chloride (30 ml).
) in a 25% hydrobromic acid/vinegar solution at 0°C;
5ml) was added and stirred for 4 hours under nitrogen gas atmosphere.
After the reaction was completed, the mixture was placed in ice water to remove hydrobromic acid, extracted with dichloromethane, and further dried over anhydrous sodium sulfate, and dichloromethane was distilled off. The obtained crude product was immediately dissolved in dry methanol, silver carbonate was added thereto, and the mixture was stirred at room temperature for 24 hours.
The studs were removed and the methanol was subsequently distilled off. The obtained crude product (1.22 g) was purified using a column to obtain N,N'-dibenzyloxycarbonyl-3,4,6,1',3'
, 6'-hexa-O-acetyl-D-gelcosamino-(1→4)-β-0-glucosamine methyl glycoside (0.52 g) was obtained. Methanol (30 ml) was added to this compound (1.0 g), stirred to form a suspension, saturated sodium carbonate (3 ml) was added, and the mixture was allowed to react for 24 hours, followed by extraction with ethyl #acid.

N'-dibenzyloxycarbonyl-0-gelcosamin (1→4)-β-D-glucosamine methyl glycoside (0.45 g) was obtained.

Subsequently, this compound (0.3 g) was added to pyridine (5 ml).
While stirring under a nitrogen gas atmosphere, add balmitoyl chloride (0.29 g; 0.3 ml) and add O.
The reaction was allowed to proceed for 12 hours at a temperature of .degree. C. to room temperature.

After the reaction was completed, 10% hydrochloric acid solution was added and extracted with ethyl acetate.The extract was further washed with saturated brine, and the solvent was distilled off. After this, remove the ethyl acetate.

A crude product (0.58 g) was obtained and further purified by column to give N,Hl-dibenzyloxycarbonyl-6°6'
-G-O-valmitoyl-〇-gelcosamin (1→4
)-β-D-glucosamine methyl glycoside (0.1g
) was obtained. This compound (0.16 g) was mixed with a mixed solvent (tetrahydrofuran:methanol = 1:1 v/v
, 10 ml) and reacted with hydrogen gas at room temperature for 12 hours. The solvent was distilled off to give the crude product (0,11
g) was obtained. Furthermore, the final product 6,6'-di○-valmitoyl-D-gelcosamino-(1→4)-β-D-glucosamine methyl glycoside (0.02 g) was obtained by pre-T LC. Yield 16.7% MS (FAB); 831(呵÷1)4I R(
KBr): 3420cm-', 2920c
m-', 2850 cm-'.

1740c■-', 1470cm-'Reference Example 6 3.6-Di-0-lauroyl-〇-glucosamine methyl glycoside production method D-N-benzyloxycarbonyl-〇-glucosamine methyl obtained from glucosamine hydrochloride according to a known method Glycoside (Log: 30.58 mmol) was dissolved in pyridine (60 ml). Add lauroyl chloride (14.71 g; 67.3 mmol) at room temperature and
The mixture was stirred for an hour, 10% hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The extracted ethyl acetate layer was washed with saturated brine, and the ethyl acetate was distilled off to obtain a crude product (19.3 g), which was further purified by IF using a column to obtain N-benzyloxycarbonyl-3,
6-di-0-lauroyl-D-glucosamine methyl glycoside (2.7 g) was obtained. This chemical compound (2.7g; 3
．． 14 mmo 1) was dissolved in a mixed solvent of methanol, 2 Wl and ethyl = 1: 1 v/v, Pd-C as a catalyst was added and reacted with hydrogen gas for 10 hours. The catalyst was removed and the crude product (1,85 g
) was obtained. 3,6-G0 by making W in a column
-Lauroyl-〇-glucosamine methyl glycoside (0
, 5g) was obtained.

m, p,: 50-54°C IR(KBr): 3380cm-', 2
940cm-', 2860cm-'1730cm-'
, 1580cm-', 1470cm-'1190c
m-', 1100cm-', 106106Otomi'H
-NMR(CDCla)δ(ppm): 0.88(t, J=6.
14.68): 1.26 (s, 3611); 1.60-
1.66 (m, 48): 2.09 (s.

3H); 2.31-2.44 (!, 2H); 2.83
~2.90 (m, 111); 4.26 ~ 4.49 (1,
11t); 4.74-4.99 (+a, IFI); 3.
35-3.45 (m, 4B); 3.77-3.79 (w
+, 1 old elemental analysis: CHN Actual PI value 66.40z 10.81% 2.32
% calculation value' 66.78 calculation 10.59% 2.5
1% (calculated value as “Cz+HseOvN”) Reference example
7 Method for producing 3.6-di-0-myristoyl-0-glucosamine methyl glycoside The title compound was obtained in the same manner as in Reference Example 6, using myristoyl chloride instead of lauroyl chloride.

m, p, : 47-50℃ IR(KBr) : 3380c
m-', 2940cm-', 2860c+e-
'1730cm-', 1580cm-', 14
70cm-'1190cm-', 1100c+s-
', 106106O'IH-NMR (C: DCl,
) δ (p p m ): 0.8 g (t, J=8.1
8,6H): 1.26 (s, 42H); 1.58-1.
70 (■, 4H), 1.87-1.93 (Sono, 2H);
2.34-2.45 (■, 4H); 2.83-2.90
(Sono, l1l); 3.36-3.45 (m, 4H); 3
．． 77-3.84 (votes, IFI): 4.26-4°52
(a, III): 4.75 (d, J=3.52, 1li
): 4.94 (t, J = 9.7.l1l).

Elemental analysis: CHN Jitsu@68.43z 11.14z 2.02%Total KM' 68.52% 10.93% 2.28%
(Calculation as “Cz s Ha t Ot N (I
Reference Example 8 3. Production method of 6-di-0-stearoyl-D-glucosamine methyl glycoside The title compound was obtained according to the production method of Reference Example 6 using stearoyl chloride instead of lauroyl chloride.

I R (K B r ): 3380c
m-', 2940ca+-', 2860cm-tomi 173
0CEl-', 1580c+a-', 1470
ca-'1190co-', 1100c+a-'
, 1060c+a-'' H-N M R (CD C
1g) δ (p p m ): 0.88 (t, J
=6.21.68); 1.25 (s, 58H), 1.5
2-1.65 (m, 411); 2.00-2.13 (s
, 2H); 2.33-2.45 (+, 4H); 2.82
~2.89 (mouth, IH), 3.35~3.55 (m, 4
II): 3.77-3.83 (c+, ill; 4.27
-4°50 (i, III); 4.75 (d, J=3.2
8.111): 4.94 (t, J=9.52,111)
-MS: 725.6155 (M
') (Ca 3 Hs a Ov N and t, r) t
txM 725.6167) In a 50 ml eggplant-shaped flask, add 100 mM phosphatidylcholine: 100 mM cholesterol: 10 mM 6-o-valmitoyl-D-glucosamine methyl glycoside dissolved in a chloroform solution at a molar ratio of 7: 2: 1. 840μl, 2407zl, 1200 each
10 ml of chloroform was added to the flask.Then, the chloroform was distilled off and vacuum dried overnight to obtain a lipid thin film on the inner wall of the flask.
5QD6 derived from bovine blood cells dissolved in 00mM sorbitol/10mM) Lis-HCl buffer 3940μm
0p1 (containing 30 Hg/ml) was added and stirred with vigorous shaking to obtain a liposome dispersion (MLV). This dispersion was left at room temperature for 2 hours in the dark, then left at about 20°C overnight, and then treated with 300mM sorbitol/10
mM) The total volume was adjusted to 12 ml with Lis-HCl buffer, centrifuged W1 (approximately 120°000 g, 70 minutes), and the supernatant was decanted to remove unencapsulated SOD, and the resulting pellet was was dispersed in 100ml of 300mM sorbitol (710mM) Lis-HCl buffer to obtain the title SOD-retaining liposome dispersion.

According to Example 1, 6-0-lauroyl- in place of 8-0-valmitoyl-〇-glucosamine methyl glycoside.
The title liposome dispersion was obtained using 〇-glucosamine methyl glycoside.

Support 5-Yu 6-0-Mi 1 through D-Lecominshi
Ibosome According to Example 1, the title liposome dispersion was obtained using 6-0-myristoyl-D-glucosamine methyl glycoside instead of 6-0-balmitoyl-D-glucosamine methyl glycoside.

According to Example 1, the title liposome dispersion was obtained using 6-0-stearoyl-D-glucosamine methyl glycoside instead of 6-0-valmitoyl-D-glucosamine methyl glycoside.

According to Example 1, instead of 6-0-valmitoyl-〇-glucosamine methyl glycoside, 6,6'-J-0-pa,lumitoyl-D-gelcosamin-(1→4)-β-
A posome dispersion described in a was obtained using D-glucosamine methyl glycoside.

According to Example 1, the title liposome dispersion was obtained using 3,6-di-0-lauroyl-D-glucosamine methyl glycoside F instead of 6-0-valmitoyl-〇-glucosamine methyl glycoside.

According to Example 1, the title liposome dispersion was obtained using 3,6-di-0-myristoyl-D-glucosamine methyl glycoside instead of 6-○-valmitoyl-D-glucosamine methyl glycoside.

According to Example 1, the title liposome dispersion was obtained using 3,6-di-O-balmitoyl-0-glucosamine methyl glycoside instead of 6-0-valmitoyl-D-glucosamine methyl glycoside.

According to Example 1, 3,6-di-0-stearoyl-D-glucosamine methyl glycoside was used in place of 6-0-valmitoyl-D-glucosaminomethyl glycoside F to obtain the title liposome dispersion.

According to Example 1, the title liposome dispersion was obtained using 6-0-valmitoyl-D-glucosamine in place of 6-0-valmitoyl-〇-glucosamine methyl glycoside.

[Test example]

Next, we will show a test example to investigate the effect of this liposome.
In addition, the abbreviations used below have the following meanings.

PC: Phosphatidylcholine Chol: Cholesterol Glu: 6-0-valmitoyl-D-glucosamine methyl glycoside Method: By quantifying the SOD activity in the supernatant in Example 1 by the nitrous acid method, the amount of SOD in the liposome was determined. The amount of SOD that was not encapsulated was determined, and this was compared with the amount of SOD added during the preparation of the liposomes to determine the amount of SOD added into the liposomes.
The OD inclusion rate was determined.

Also, for comparison, Pc that does not contain glucosamine derivatives
: Chol: 8: A neutral liposome dispersion consisting of 2 (molar ratio) was similarly prepared and its encapsulation rate was determined.

The results are shown in Table 2-1.

Table 1 As is clear from the above results, cationic liposomes containing glucosamine derivatives showed a higher drug encapsulation rate than neutral liposomes not containing glucosamine derivatives.

2 Rest - Method: DDY male rats were anesthetized with urethane, 500 μl of the 3H-labeled liposome dispersion prepared according to Example 1 was intravenously injected into the femoral vein, and blood was withdrawn from the carotid artery over time. Collected into an Eppendorf tube. This was centrifuged, the supernatant was taken into a 100 μl vial, 700 μl of Solnin 350 was added and dissolved, and 2N hydrochloric acid 180 μl was added.
Neutralized with μl. Furthermore, add 6 ml of Clearsol and place in a liquid scintillation counter (LS 5000TA).
Radioactivity was determined using BECKMAN).

As a control, a liposome dispersion with 3H-labeled Pc:Chol=3:1 (molar ratio) was prepared, and the radioactivity was similarly measured to study the pharmacokinetics.

In addition, based on these results, the respective blood concentrations (μ
g/ml), time curve underside ratio (AUG: blood concentration x time (hr)), and blood half-life (tl/2: hr).

In addition, the 3H-labeled liposome formulation used in this study contained a trace amount (17
500,000 mol) and prepared according to Example 1.

The results are shown in Table 2.

Table 2 As is clear from the above results, the AUG of liposomes containing glucosamine derivatives as a membrane material is increased by about 4.1 times, and t, 7□ is increased by about 2.5 times compared to liposomes that do not contain glucosamine derivatives. It was extended to

Method: As in Test Example 2, cationic lyssomes containing H-1111 were intravenously injected into the femoral vein of rats anesthetized with urethane. After 8 hours, the whole blood of the rats was collected, and each organ was removed. Then, each radioactivity was measured using a liquid scintillation counter, and the distribution status of the gluegosomes in each organ was determined as aFJ.

Conclusion! #: Shown in Table-3.

(Left below) Table 3 (n=3) As is clear from the above results, glucosamine derivatives
Compared to neutral liposomes that do not contain cationic liposomes, the amount of cationic liposomes transferred into each organ is smaller. It is a liposome with excellent targeting properties.

4 Now, method: To what extent is the toxicity of the cationic liposome of the present invention containing a glucosamine derivative as a membrane material compared to the conventionally known cationic liposome containing stearylamine as a membrane material? To find out the mmt of both liposomes without encapsulating any drug, a lethal toxicity test was conducted on mice.

1. mW of test solution (1) Liposome dispersion containing 6-0-valmitoyl-D-glucosamine methyl glycoside. - A liposome dispersion containing glucosamine methyl glycoside as a membrane material was obtained. This was concentrated using an ultrafiltration membrane, and further diluted with sterile distilled water for injection as necessary to obtain a test solution. Phosphorus in the same test solution was quantitatively analyzed by the ammonium molybdate method, and the concentration of 6-0-balmitoyl-〇-glucosamine methyl glycoside was determined from the blending ratio with phosphatidylcholine.

(2) Stearylamine-containing liposome dispersion Example 1
A liposome dispersion containing stearylamine as a membrane material was obtained using the method described above, but without adding a solution of the drug SOD. This was concentrated using an ultrafiltration membrane, and if necessary, diluted with sterile distilled water for injection to obtain a test solution. Phosphorus in the same test was quantitatively analyzed using the ammonium molybdate method, and the stearylamine concentration was determined from the blending ratio with phosphatidylcholine.

2. Test method ICR &! Sex mice were divided into groups of 2 to 3 mice, and 2.0 ml of each test solution was administered once at JI [intracavitally]. The solvent control group received 2.0 ml of sterile distilled water.

After administration of the test solution, the general condition of the animals was carefully observed at least once a day for 16 days, and signs of growth and mortality were recorded.

3. Test results shown in Table 2-4 The test substance is as shown below.

■Posome dispersion containing 6-0-valmitoyl 〇-glucosaminomethyl glycoside■Liposome dispersion containing stearylamine (blank below) Table 4 Quantity as Cyto or stearylamine.

In the above test, the 6-0-valmitoyl-
Regarding the dispersion of cationic liposomes containing 〇-glucosamine methyl glycoside as a membrane material, there were no deaths during the observation period, and the LD5eW was 1154.8 m as 6-0-valmitoyl-〇-glucosamine methyl glycoside.
It is estimated that it is more than g/kg. On the other hand, in the case of a known cationic liposome dispersion containing stearylamine as a membrane material, death cases were observed even at low doses, and the LD, s value was 157.9 mg/kg (146.0 to 170°8
mg/kg).

From the above results, it can be said that the cationic liposome containing the novel glucosamine derivative of the present invention as a membrane material is a highly safe cationic liposome with extremely low toxicity compared to conventionally known cationic liposomes.

(Effects of the Invention) The cationic liposomes obtained by the present invention have extremely low toxicity compared to conventional cationic liposomes containing stearylamine, and the encapsulation rate of anionic or neutral drugs is within the normal range. It has a higher potency than anionic or anionic liposomes, has a longer blood circulation period, and is less likely to migrate into organs.Therefore, this liposome can be applied not only to SOD but also to other physiologically active substances. , anti-inflammatory agents, hormonal agents, anti-cancer agents, and other pharmaceutical applications.In particular, when SOD is encapsulated, it can be used to treat various diseases (e.g., chronic inflammatory diseases, heart and brain diseases caused by ischemia-reperfusion injury, digestive disorders, etc.). It is considered to be effective for the treatment of organ diseases, etc.

Claims (1)

[Claims] 1. General formula ▲ Numerical formula, chemical formula, table, etc. ▼ [I] (In the formula, R^1 and R^2 are hydrogen atoms or -CO(CH_
2) _nCH_3 group (n means an integer of 10 to 22), R^3 means a hydrogen atom or a lower alkyl group, and m means an integer of 0 to 3. However, R^1, R
^2 cannot be a hydrogen atom at the same time. ) A liposome containing a glucosamine derivative or a salt thereof as a membrane material. 2. The liposome according to claim 1, which contains a polypeptide having superoxide dismutase activity. 3. Glucosamine derivative is 6-O-palmitoyl-D-
The liposome according to claim 1 or 2, which is glucosamine methyl glycoside.