JPS63313724A - Sialic acid modifying polysaccharide coated ribosome - Google Patents

Abstract

PURPOSE:To obtain a ribosome coating body significantly reduced in phagosome number and lowered in vanishing speed of the ribosome in blood, by coating ribosome membrane surface containing ubidecarenone with a specific modifying polysaccharide derivative. CONSTITUTION:0.5-5.0 sugar unit per 100 sugar unit constituting pullulan, amylose, amylopectin, dextrane and/or mannan originates in a derivative obtained by substituting primary hydroxyl group in carbon at 6-position thereof by formula I (R is H or cholesteryloxycarbonyl). The sugar unit when R is cholesteryloxycarbonyl is 0.5-4.5 and 0.5-10.0 sugar unit per 100 sugar unit constituting the derivative is a sialic acid-modifying natural substance-derived polysaccharide derivative wherein primary hydroxyl group in the carbon at 6-position thereof is further substituted by a sialic acid residue expressed by formula II and the above-mentioned derivative is applied to membrane surface of ribosome containing ubidecarenone to provide the aimed product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a liposome-coated body. More specifically, the present invention relates to a liposome-coated body formed by coating the membrane surface of a ubidecarenone-containing liposome with a specific modified naturally occurring polysaccharide derivative.

In recent years, in the fields of medicine and pharmacy, organs used for drugs,
There is growing interest in so-called target-directed drugs that efficiently transfer drugs to cells, maximize drug efficacy, and minimize side effects.

As one means for achieving this objective, technology that utilizes liposomes as carriers has come to be regarded as important. As an invention of such technology, for example, Japanese Patent Application Laid-open No. 52
-143.218, JP 52-15L718, JP 53-133.616, JP 55-8.488
Inventions in each publication can be cited.

On the other hand, ubidecarenone, also known as Coenzyme Q.

has recently come into widespread clinical use as a drug effective for improving cardiac function. By incorporating this ubidecarenone into liposomes, organ migration can be increased (Japanese Patent Application Laid-Open No. 58-8.010,
8-201,711 and JP-A-60-1,124. ) The liposomes used as carriers of ubidecarenone are phagocytosed in large quantities by the phagocytic cells present in the reticuloendothelial threads after in vivo administration, and disappear from the blood very quickly. Further, improvements are needed when increasing the efficiency of transfer to specific organs or cells, or when used for diseases where the lesion is located within the blood vessels.

In view of the difficulties involved in achieving 6', the present inventors have proposed that sialic acid residues on the surface of red blood cell membranes play an important role in evasion of red blood cells from phagocytic cells and long-term circulation in the bloodstream. We paid attention to the fact that it was effective and conducted extensive research. As a result, the desired solution was successfully achieved by coating the surface of the ubidecarenone-containing liposome membrane with a specific modifier II derivative described in detail below.

That is, as a result of measuring the amount of phagocytosis of ubidecarenone-containing liposomes to human phagocytes, the actual amount of phagocytosis was significantly reduced when the liposome membrane surface was coated with a specific modified polysaccharide derivative. Furthermore, when the liposomes were intravenously administered to animals, the rate of disappearance from the blood was significantly lower than that of ordinary liposomes not coated with modified polysaccharide derivatives.

Therefore, the present invention provides that 0.5 to 5.0 sugar units per 100 sugar units constituting pullulan, amylose, amylopectin, dextran and/or mannan have a primary hydroxyl group at the 6-position carbon of the formula %. % (wherein R represents H or a cholesteryloxycarbonyl group), and when R is a cholesteryloxycarbonyl group in the formula, the number of sugar units is 0.5 to 4.5 Naturally derived A polysaccharide derivative (so-called primary modified naturally derived poly-1i derivative) comprising 10 sugar units constituting it.
0.5 to 10.0 sugar units per sugar unit are sialic acid-modified naturally derived multi-W! The object of the present invention is to provide a liposome-coated body comprising a membrane surface of a ubidecarenone-containing liposome coated with a derivative (so-called second-order modified naturally-derived polysaccharide derivative).

In this case, the secondary modified naturally occurring polysaccharide derivative may be used alone or in the form of a mixture of two or more types.

The polysaccharide derivative in the present invention will be explained as follows.

In other words, in the naturally occurring polysaccharides pullulan, amylose, amylopectin, dextran and/or mannan, 0.5 per 100 sugar units constituting it.
The primary hydroxyl group at the 6-position carbon of ~5.0 sugar units is an aminoethylaminocarbonylmethyl group (hereinafter abbreviated as rAEcMJ) represented by the formula % (wherein R represents H or cholesteryloxycarbonyl group) ) (i.e., the first modified naturally occurring polysaccharide derivative). In this case, the number of sugar units in which R is a cholesteryloxycarbonyl group is 0.5 to 4.5. The cholesteryloxycarbonyl group (hereinafter abbreviated as "rchoffi") is represented by the following structural formula.

The sialic acid naturally-derived polysaccharide derivative (that is, the second modified naturally-derived polysaccharide derivative) in the present invention is this first-modified naturally-derived polysaccharide derivative, and the saccharide unit 1 constituting it is
The primary hydroxyl group at the 6-position carbon of 0.5 to 10.0 sugar units per 00 sugar units is further substituted with a sialic acid residue (hereinafter abbreviated as rNeuN^) represented by the formula.

The sialic acid-substituted naturally occurring polysaccharide derivative used in the present invention can be produced by a conventionally known method using sialic acid as a starting material. For example, the outline is as follows.

Sialic acid is reacted with acetyl chloride, and then reacted with hydrogen chloride gas to obtain a chlorinated product [0Ac-CIt-NeuNA:l] of sialic acid protected by an acetyl group.

This was mixed in a mixed solvent of anhydrous DMSO and anhydrous DMF.
Next modified naturally derived polysaccharide derivative (Cho l -AECM-
polysaccharide] and a silver trifluoromethanesulfonate catalyst, and then deprotected in an aqueous sodium hydroxide solution, a secondary naturally occurring polysaccharide derivative (Ch
o I2-AECM-polysaccharide-NeuNA) is obtained.

The reaction steps for glucobylanose units having NeuNA groups are as follows.

Chol(1,9)-8ECM(1,2)-70Luran(50)Chol(1,9)-AECM(2,1)-Pullulan(50)-NeuNA(3,8) Typically, pullulan The modified products are as follows, taking amylopectin and amylopectin as an example.

ChoA (1,9)-AECM(2,1)-pullulan(50)-NeuNA(3,8) This has 1.9 cholesteryloxycarbonyl groups (ChoI!,) per 100 glucobylanose units. In pullulan with an average molecular weight of 50,000 and substituted with 2.1 aminoethylaminocarbonylmethyl groups (AECM), 3.8
This is the case where 3 sialic acid residues (NeuNA) are substituted.

The 'H-NMR spectrum of this product is shown in FIG. 1, and the IR spectrum is shown in FIG.

Cho l (1,8) -AECM (3,4)-Amylopectin (1,12) -NeuNA (4,1) This is 1.8 Cho l. per 100 glucobylanose units. In amylopectin with an average molecular weight of 112,000 substituted with 3.4 AECMs, 4
．． This is the case where one NeuNA is substituted.

The '+1-NMR spectrum of this product is shown in FIG. 3, and the IR spectrum is shown in FIG.

In the liposome of the present invention, Evidekallumen is contained in the membrane that constitutes the liposome. The basic substances constituting the liposome membrane are mainly phospholipids and sterols, and ubidecarenone coexists with these and is uniformly dispersed in the membrane. Examples of the phospholipids used in the present invention include phosphatidercholine,
Mention may be made of phosphatidylethanolamine, phosphatidylserine, sphingomyelin, stearylamine dicetylphosphate, phosphatidylglycerol, phosphatidylinositol phosphatidate, and mixtures thereof. However, it is not limited to these.

Cholesterol is the most preferred sterol.

Regarding the composition ratio of ubidecarenone, phospholipids, and sterols, 1 molar ratio of ubidecarenone to 1 phospholipid
It is enough if the molar ratio is 0 or more, and the sterol is 1 mole higher.
. For example, Yu, Aka.

1 molar ratio, when egg yolk phosphatidylcholine is used as the phospholipid, the molar ratio is 10 to 30, and when cholesterol is used as the sterol, the molar ratio is 1 to 10.
The molar ratio is the preferred range of use.

The liposomes used in the present invention are spherical particles with a particle size of about 0.1 to 5.0 μm when viewed under an electron microscope.

The ubidecarenone-containing liposome used in the present invention can be produced by applying conventional techniques mutatis mutandis.

For example, liposomes and ubidecarenone are placed in an eggplant flask, and chloroform is added to dissolve them once. Next, the solvent is distilled off, and the resulting film is peeled off by adding glass beads and a suitable buffer solution. After this is sonicated, it is passed through a Sephadex or Sepharose column to collect the liposome fractions. It may also be used as is without column treatment.

In order to coat the ubidecarenone-containing liposome with the second modified naturally-derived polysaccharide derivative of the present invention, the dispersion of the modified polysaccharide derivative is added to a suspension of the ubidecarenone-containing liposome and stirred. For coating, only one type of modified polysaccharide derivative may be used, or a mixture of two or more types of modified polysaccharide derivatives may be used.

Next, the present invention will be explained in more detail with reference to Examples.

Example I CoQ + by ChoIl(1,9)-A[ICM(2,1)-pullulan(50)-NeuNA(3,8). Production of liposomes with 4-containing liposomes Egg yolk phosphatidylcholine: 60■, Cholesterol: 9.75■
and ubidecarenone or C00,.

6.3■ was dissolved in 4 d of chloroform in an eggplant-shaped flask, and the chloroform was removed under reduced pressure using a rotary evaporator. The obtained thin film was dried overnight in a vacuum desiccator to completely remove chloroform. Apply 2 times of 2.0 d to the thin film remaining at the bottom of the eggplant-shaped flask, for a total of 4
Add phosphate buffer (pH 7.4) and 1 glass bead and incubate the Portex until the membrane is completely peeled off.
Shake it with a mixer.

Next, transfer this suspension to a test tube and place it under a stream of nitrogen.
After intermittent sonication using a sonicator at 5°C, a yellow-white suspension was obtained. CoQ in this suspension
,. Contains liposomes.

Separately, pullulan into which sialic acid residues and cholesteryloxycarbonyl groups have been introduced (molecular weight 50,000, degree of sialic acid substitution 3.8, degree of cholesteryloxycarbonyl group substitution 1.9) was added to a phosphate buffer solution (pl(7,4 ) was mixed to prepare a dispersion liquid (6.7 mg 10.44 m).

The yellow-white suspension 1.33 mI! The above dispersion was added to the mixture and stirred at room temperature for 30 minutes to obtain a coated body.

Example 2 C00 with Chop (1,8)-AECM(3,4)-Amylopectin(112)-NeuNA(4,1)
．． . In the description of Example 1, in place of pullulan into which a sialic acid residue and a cholesteryloxycarbonyl group were introduced,
Same as Example 1 except that amylopectin into which sialic acid residues and cholesteryloxycarbonyl groups were introduced (molecular weight 112,000, degree of sialic acid substitution 4.1, degree of cholesteryloxycarbonyl group substitution 1.8) was used. A liposome-coated body containing ubidecarenone was obtained.

Example 3 Ubidecarenone-containing liposome coatings were obtained in the same manner as in Example 1, except that modified amylose, modified dextran, or modified mannan was used instead of modified pullulan.

Example 4 A ubidecarenone-containing liposome-coated body was obtained in the same manner as in Example 1, except that modified pullulan into which no cholesteryloxycarbonyl group had been introduced was used.

Example 5 Same as Example 4, but instead of modified pullulan in which no cholesteryloxycarbonyl group was introduced,
Ubidecarenone-containing liposome coatings were obtained using modified amylopectin, modified amylose, modified dextran, or modified mannan into which no cholesteryloxycarbonyl group was introduced, respectively.

The effect of coating ubidecarenone-containing liposomes with modified polysaccharide derivatives will be explained below using experimental examples.

Experimental Example I
PPC) 4 liposomal egg yolk phosphatidylcholine 80 μl was dissolved in chloroform 4 in an eggplant-shaped flask, and the chloroform was removed under reduced pressure using a rotary evaporator. The obtained thin film was dried overnight in a vacuum desiccator to completely remove chloroform. The thin film remaining on the bottom of the eggplant-shaped flask was peeled off with 4 m of physiological saline using a portex mixer. Next, this suspension was transferred to a test tube with branches, and subjected to ultrasonication for 5 minutes using a probe-type sonicator at 0°C under a nitrogen stream to form liposomes.

This liposome suspension was added to a thin film of 10 μCi of (+4c)-dipalmitoylphosphatidylcholine (DPI), C), which was prepared separately in a manner similar to that described above.

After this thin film was peeled off using a portex mixer, labeled multilamellar liposomes were fractionated and collected using a Sepharose 4B column.

To this (14G)-DPPC-labeled multilamellar liposome, the following modified polysaccharide derivatives were respectively added and incubated at 20°C for 1 hour.

(Unexamined Japanese Patent Publication No. 60-1,124, Unexamined Japanese Patent Publication No. 58-201.7
See the coating notes in each publication of No. 11. )■ Chop
(1,9)-AECM(2,1)-pullulan (50)
-NeuNA (3,8), ■ Chop (1,9) -AECM (2,1) - Pullulan (50), ■ Choj2 (1,8) -AECM
(3,4)-Amylopectin (112)-NeuNA
(4,1), ■ ChoI! , (1,8)-AECM(
3,4)-Amylopectin was further diluted with physiological saline to 2.0 x 10'' striped stratified liposomes (150 μl)□.

b. Human and Human Cells Monocytes and neutrophils were isolated from 100 type A blood by centrifugation in a conventional manner. And 2 each
．． 6X 10'cell/450. # j2 RPM
I-1640 using a culture solution (R containing 10% FC5)
PMI-1640).

B. Method 1011! i! In a test tube, 2. OX 10” striped stratified liposome 150ttl and 2.OX 106cell
/450μI! and cell suspension (monocytes or neutrophils) to make a total of 500 μl. This was placed in a constant temperature bath at 37°C.
The cells were incubated with shaking for 1 hour to allow the cells to phagocytose the liposomes. Added 500 μl of silicone oil 2
000μ! Transfer this cell suspension to the sample containing the
Only the cells were removed by centrifugation at 1000 x g, and each radioactivity of the supernatant and cells was measured using a liquid scintillation counter. The number of phagocytic vacuoles of various liposomes injected into human monocytes and human neutrophils per cell was calculated from the ratio of the radioactivity of the cells alone to the total radioactivity of the cell and the cell. These are shown in FIGS. 5 and 6, respectively.

(Average value of 3 cases). In addition, uncoated liposome ■ is also shown as a control.

From these figures, it can be seen that the multilamellar liposomes coated with 3 are less susceptible to phagocytosis than the multilamellar liposomes coated with .

It is also seen that multilayer liposomes coated with ■ are significantly less easily phagocytosed than liposomes with ■.

Experimental Example 2 A, Sample Samples x - z below were prepared in the same manner as in Examples 1 and 2, except that "C-Co" was used instead of CoQ 1° in the description of Sample Examples 1 and 2. A sample was prepared.

X, 14C-CoQ+o-containing liposome 3', "C
-CoQ+. Containing liposome-coated body, except that the coated body used was the one in Example 1.

z, 14C-Co mouth. Containing liposome-coated body, except that the coated body used was the one in Example 1.

Note that 14C-1cOQII+ is radiolabeled ubidecarenone represented by the following structural formula.

*14C-label position Also, this specific radioactivity is 7.95μCi/■, and radiochemically, two types of developing solvents [chloroform/benzene-171, acetic acid/benzene-1/9 (volume ratio)] It was found to be single by thin layer chromatography using .

B. Animal experiment: A sample of 0.97 cm C-CoQ+/kg was poured into the left femoral vein of a male guinea pig (weight 240 to 270 g) and sutured. After that, the guinea pig was left in the breeding cage. Blood was then collected from the ear vein at predetermined intervals. The +4C-CoQ++ concentration in the collected blood was measured by the method described below.

Measurement of radioactivity in blood Collect 20 μl or 50 μl of blood from the ear vein, and
．． Solubilize with 75-related 5oluene 350/isopropyl alcohol (1/1, volume ratio), decolorize by adding a few drops of hydrogen peroxide, instagello, 5NhC
1 (9/1 volume ratio) was added, and radioactivity was measured using a liquid scintillation counter.

14C-CoQl when administering C0 fatty acid sample. Figure 7 shows the time course of radioactivity in the blood. In this figure, the O-marked line, the *-marked line, and the Δ-marked line indicate the changes (average value of three cases) when Sample X, Sample y, and Sample 2 were administered, respectively.

As shown in FIG. 7, coating with ■ or ■ reduces the rate at which liposomes disappear from the blood.

As shown in the above experimental examples, ubidecarenone-containing liposomes coated with the second modified naturally occurring polysaccharide derivative of the present invention significantly reduced the number of cysts and significantly decreased the rate of disappearance of the liposomes from the blood. decrease to.

[Brief explanation of the drawing]

Figure 1 shows the 'H-
The NMR spectrum is shown. FIG. 2 shows the IR spectrum of sialic acid-modified pullulan. FIG. 3 shows the ')l-NMR spectrum of sialic acid-modified amylopectin according to the present invention. FIG. 4 shows the IR spectrum of sialic acid-modified amylopectin. FIG. 5 is a graph showing a comparison of the number of cysts in human monocytes between the ubidecarenone-containing liposome itself and the ubidecarenone-containing liposome coated with the modified naturally-derived polysaccharide derivative according to the present invention. FIG. 6 is a graph showing a comparison of the number of cysts in human neutrophils between the ubidecarenone-containing liposome itself and the coated ubidecarenone-containing liposome according to the present invention. Figure 7 shows the ubidecarenone-containing liposome itself and the coated ubidecarenone-containing liposome (two types) according to the present invention.
It is a diagram showing the transition of disappearance from blood of Patent applicant: Eisai Co., Ltd. Figure 5 Figure 6

Claims (5)

[Claims] (1) 10 sugar units constituting pullulan, amylose, amylopectin, dextran and/or mannan
0.5 to 5.0 sugar units per sugar unit have a primary hydroxyl group at the 6th carbon position of the formula -OCH_2CONHCH_2CH_2NHR (in the formula R
represents H or a cholesteryloxycarbonyl group), and in the formula, when R is a cholesteryloxycarbonyl group, the number of sugar units is 0.5 to 4.5. 0.5 to 10.0 sugar units per 100 sugar units that make up the 6-position carbon are sialic acid residues whose primary hydroxyl group is further represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A liposome-coated body comprising a membrane surface of a ubidecarenone-containing liposome coated with a sialic acid-modified naturally occurring polysaccharide derivative substituted with . (2) The liposome-coated body according to claim 1, wherein the liposome membrane is a membrane composed of phospholipids and styrene. (3) The liposome-coated body according to claim 2, wherein the phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, stearylamine dicetylphosphate, phosphatidylglycerol, phosphatidylinositol phosphatidic acid, or a mixture thereof. . (4) In the ubidecarenone-containing liposome, 10 moles or more of phospholipid per 1 mole ratio of ubidecarenone,
The liposome-coated body according to claim 2 or 3, wherein the sterol is contained in an amount of 1 mole or more. (5) The liposome-coated body according to claim 2 or 4, wherein the sterol is cholesterol.