JP3085485B2 - Oligosaccharide derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a newly synthesized oligosaccharide derivative having a well-defined structure, which is useful for imparting stability to phospholipid vesicles (hereinafter referred to as "vesicles"). The endoplasmic reticulum can contain a physiologically active substance such as adriamycin or prostaglandin in its inner aqueous phase and can be used as a drug delivery system or sustained-release material, and can also contain purified hemoglobin to form artificial erythrocytes. Can also.

[0002]

2. Description of the Related Art The endoplasmic reticulum is agglomerated or fused, or
It is normal to have instability such as a change in form and leakage of inclusions due to the above phenomenon. Many proposals have been made as means for solving such instability. First, a solution by polymerization of an aggregate of a phospholipid having an unsaturated group has been attempted (for example, a review: H. Ringsdorf).
t al. Angew. Chem. , Int. Eng
l. , 27, 113 (1988)), the properties of lipids, such as gel-liquid crystal phase transition, phase separation and fluidity of the membrane, are lost due to the membrane polymerization, and the functions related to these are lost. Loss occurs.

[0003] Next, the surface coating of the endoplasmic reticulum with carboxymethylchitin (H. Izawa et al., Bio).
chim. Biophys. Acta, 855, 243
(1986)) or coating the surface of the endoplasmic reticulum with the compound in which a hydrophobic group such as cholesterol is bonded to a polysaccharide such as dextran or pullulan (J. Sunamoto et al.).
l. , J. et al. Biochem. , 88 1219 (198
0)), attempts have been made to stabilize the structure of the endoplasmic reticulum. However, these do not significantly change the membrane fluidity of the endoplasmic reticulum, but have the drawback of easily inducing aggregation.

[0004] When an amphipathic molecule in which polyethylene glycol is bound to the hydrophilic part of cholesterol or phosphatidylethanolamine is introduced into the endoplasmic reticulum, the aggregation of the endoplasmic reticulum is suppressed and stabilized, and the endoplasmic reticulum is retained in the blood. It has been reported that time can be extended (TM Allen)
et al. , Biochim. Biophys. Ac
ta, 1066, 29 (1991)).

The present inventors also disclose that the introduction of the oligosaccharide fatty acid ester developed by the present inventors into the endoplasmic reticulum bilayer membrane is extremely effective for stabilizing the dispersion of the endoplasmic reticulum. (JP-A-1-294701). In that case, an ester derivative having a longer sugar chain shows a better effect, and it is necessary to use an oligosaccharide rather than a monosaccharide or a disaccharide.

[0006]

Although the invention disclosed in the above publication provides many solutions to the stability of the endoplasmic reticulum, long-chain fatty acids to oligosaccharides having a large number of reactive groups (hydroxyl groups) can be obtained. The introduction of the group is carried out non-selectively, and the number and site of introduction are various. Therefore, the separation and purification are complicated and the yield is low, and the effect of stabilizing the dispersion state of the endoplasmic reticulum and the difficulty of immobilizing the oligosaccharide fatty acid ester on the endoplasmic reticulum membrane surface differ depending on the isomer of the oligosaccharide. Is the problem. further,
The relationship between the structure of the isomer and the effect is also unknown. Therefore, in the present invention, in order to solve the above-mentioned problem, it is an object of the present invention to develop an oligosaccharide derivative having a clearly defined structure in which a hydrophobic group is selectively introduced into a specific group of the oligosaccharide.

[0007]

In order to solve the above-mentioned problems, an object of the present invention is to provide an oligosaccharide having a structure and effect clearly obtained by selectively introducing a hydrophobic group at the anomeric position of the oligosaccharide. It is concerned with providing derivatives. That is, the present invention has a sugar polymerization degree of 3 to
The present invention relates to an oligosaccharide derivative represented by an integer of 20 and having a hydrophobic group introduced at an anomeric position of a reducing terminal group through an ether bond.

Hereinafter, the present invention will be described in detail. Since the oligosaccharide derivative of the present invention is a compound that needs to adjust the hydrophilic-hydrophobic balance, the oligosaccharide used in the present invention is, for example, maltopentaose,
The above-mentioned balance adjustment is easy, the sugar polymerization degree is 3-20.
The oligosaccharide represented by the following integer is preferable, and the reducing end group is preferably an oligosaccharide composed of aldose, and a sugar alcohol of the oligosaccharide is also included.

The oligosaccharides having a degree of sugar polymerization represented by an integer of 3 to 20 can be obtained in a high purity by fractionating the oligosaccharides by the degree of polymerization or the type by using gel filtration while keeping the column warm. A differential refractometer is used for the detection. The qualitativeness and purity of the separated oligosaccharides were confirmed using a mixed solvent (1) using a silica gel thin plate.
-Butanol / pyridine / water) to develop color with sulfuric acid, or liquid chromatography using an amino column as a stationary phase and a mixed solvent (acetonitrile / water) as a mobile phase. The oligosaccharide used in the present invention may be a commercially available pure product as long as the structure is clear.

In the oligosaccharide derivative of the present invention, since it is necessary to pay attention to the adjustment of the hydrophilic-hydrophobic balance, the hydrophobic group used here is, for example, an alkyl group having 12 carbon atoms.
-22, and the alkenyl group preferably has 12-22 carbon atoms and the unsaturated number is 1-4. An alkenyl chain refers to the case of one alkenyl group and the case of glycerol in the form of a dialkylglycerol having two alkenyl groups at the 1,2- or 1,3-positions of glycerol. Examples of the linear alkyl group having 12 to 22 carbon atoms include lauryl group, cetyl group, stearyl group, behenyl group, and octadecyl group. Examples of the alkenyl group having 12 to 22 carbon atoms include 1,1-dodecenyl. And a branched alkenyl group in addition to a linear alkenyl group such as a group, a 1-oleyl group and a linolenyl group. These alkenyl groups are the 1,3 groups of glycerin.
It can also be used in the form of a dialkenyl glycerol having a structure linked to the 2- or 1,2-position by an ether bond.

The phospholipid vesicle dispersion stabilizer and the phospholipid composition for producing vesicles of the present invention have a sugar polymerization degree of 3
The hydrophobic group in the oligosaccharide derivative represented by an integer of ２０20 and having a hydrophobic group introduced at the anomeric position of the reducing terminal group by an ether bond is an alkyl chain having 12 to 22 carbon atoms, and an unsaturated bond having 1 to 4 carbon atoms. The alkenyl chain preferably has 12 to 22 carbon atoms. In the "alkyl chain" and "alkenyl chain", only one alkyl group or alkenyl group is ether-bonded to the anomeric position of the reducing terminal group of the oligosaccharide. In addition to the above case, glycerol has two alkyl groups or alkenyl groups bonded to the 1,3-position or 1,2-position of glycerin, but the glycerol residue is ether-bonded to the anomeric position of the reducing end group of the oligosaccharide. Cases are included. When these hydrophobic groups are exemplified including an ether bond, there are primary alcohol and secondary alcohol residues having 12 to 22 alkyl carbon atoms. That is, examples of the linear primary saturated alcohol include lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, and the like. In addition, linear primary unsaturated alcohols such as 1,1-dodecenol, oleyl alcohol, and linolenyl alcohol And alcohol residues of branched primary saturated alcohols, branched primary unsaturated alcohols, secondary saturated alcohols and secondary unsaturated alcohols. In addition, 1,3-glycerin
And a dialkylglycerol residue bonded to the 1- or 1,2-position with the above-mentioned primary saturated alcohol and primary unsaturated alcohol. Further, alcohol residues such as sterols such as cholesterol, cholestanol, sitosterol and ergosterol are also included in the scope of the hydrophobic group.

The introduction of a hydrophobic group to the terminal anomeric position of a sugar chain by an ether bond is carried out by a method in which dialkyl glycerol is ether-bonded to the terminal anomeric position of a disaccharide (T. Ogawa et al., Carbohydrr).
ate Res. 93, C6 (1981); Oga
wa et al. , Agric. Biol. Che
m. , 45 (10), 2392 (1981); O
gawa et al. , Agric. Biol. Ch
em. , 46 (1), 255 (1982)).

First, protection of the hydroxyl group of the oligosaccharide with an acetyl group is performed by adding distilled pyridine to the oligosaccharide and then dropwise adding acetic anhydride under ice-cooling. This was left at 4 ° C. overnight, and recrystallized from isopropyl alcohol to obtain an acetyl form. The introduction of the hydrophobic group to the sugar terminal anomeric position by ether bond is carried out by using the above-mentioned hydrophobic compound having a hydroxyl group as a catalyst of trimethylsilyltrifluoromethanesulfonic acid (hereinafter, referred to as MSCF) in dichloroethane as a powder of molecular sieve 4A. In the presence, the reaction is carried out by reacting with the acetylated oligosaccharide.

Thereafter, the desired oligosaccharide derivative is obtained by removing the protecting group. Before the deprotection, the acetylated oligosaccharide derivative must be sufficiently purified. That is, the obtained acetylated product is subjected to liquid chromatography using silica gel as a stationary phase and a mixed solvent of toluene and ethyl acetate as a mobile phase. The confirmation of the fraction is performed by Fourier transform magnetic resonance measurement or infrared spectroscopy. The method for separating and purifying the acetylated oligosaccharide derivative is not limited to the above method, and a method such as supercritical chromatography, purification using a reversed-phase column, or solvent extraction can also be selected.

Finally, the acetyl group is removed by adding a basic substance such as ammonia water or triethylamine to the acetylated oligosaccharide derivative in a mixed solvent of methanol and water and refluxing for 12 hours or more. Thereafter, recrystallization is performed twice or more with a polar solvent such as methanol to obtain a final product of the oligosaccharide derivative of the present invention. Identification of the oligosaccharide derivative of the present invention can be performed by proton-NMR, carbon-NMR, or IR.

The purity of the oligosaccharide derivative obtained by the ether bond obtained as described above is confirmed by thin-layer chromatography based on silica gel or high-performance liquid chromatography.

The stability of the endoplasmic reticulum using the oligosaccharide derivative of the present invention can be confirmed as follows. Phospholipids, or cholesterol phospholipids and membrane stabilizers, cholesterol and other sterols,
Or phospholipids, sterols and stearic acid,
A hydrate is prepared by adding 0.01 to 20 mol%, preferably 0.05 to 5 mol%, of the oligosaccharide derivative of the present invention to a mixed lipid of a negatively charged lipid such as dicetyl phosphate. A vesicle dispersion of 50 to 200 nm is prepared from the hydrate by a known method such as an ultrasonic stirrer, a microfluidizer, or an extruder. The particle size of the vesicle dispersion liquid can be confirmed by a particle size distribution measuring device or a transmission electron microscope negatively stained.

The oligosaccharide derivative not introduced into the endoplasmic reticulum is removed from the dispersion of the endoplasmic reticulum by gel filtration or ultracentrifugation, and the sugar in each fraction is quantified by the phenol-sulfuric acid method. By doing so, the rate of introduction of the oligosaccharide derivative into the endoplasmic reticulum can be determined. Almost 90% or more of the oligosaccharide derivative of the present invention is introduced into the endoplasmic reticulum.
The dispersion stability of the endoplasmic reticulum can be evaluated and confirmed by measuring the change over time in the absorbance (turbidity) at 500 to 800 nm with a UV-visible spectrophotometer at room temperature for the dispersion filtered by gel filtration.

Other methods for evaluating the dispersion stability of vesicles include a method of measuring the viscosity of a dispersion of vesicles using a rotational viscometer, and a microscopic method using an electron microscope or the like.

[0020]

The oligosaccharide derivative of the present invention is suitable as a phospholipid aggregate modifier because it has a clear structure and a very good affinity for phospholipids. Furthermore, the oligosaccharide derivative introduced into the phospholipid aggregate can maintain the sugar structure exposed to the aqueous phase, and can be uniformly dispersed on the membrane of the phospholipid aggregate.

The oligosaccharide derivative as the phospholipid aggregate modifier of the present invention can not only prevent leakage of vesicle inclusions, but also, for example, stabilize dispersion of a protein or the like in a solvent, parentalization, suppression of vesicle aggregation. , Membrane fusion prevention, labeling for recognition with a specific antibody, suppression of phagocytosis in a living body, and the like, depending on the purpose of use.

When a carrier containing a functional molecule is administered to a living body in the inner aqueous phase of the endoplasmic reticulum, the longer the residence time in the blood, the higher the effectiveness. However, the endoplasmic reticulum is composed of a water-soluble polymer or It is usually aggregated by the interaction with calcium ions and the like, and is usually removed from the blood in a short time by the uptake into the reticuloendothelial system and phagocytic cells. However, the residence time of the endoplasmic reticulum in blood can be controlled by surface modification of the endoplasmic reticulum with the oligosaccharide derivative of the present invention.

Further, the oligosaccharide derivative having an unsaturated bond in the hydrophobic part of the present invention is used in combination with a polymerizable phospholipid, for example, by coating these on the surface of a culture vessel and copolymerizing the same to form a vessel surface. It is optimal for use in converting to a hydrophilic and cell-philic surface, and gives a drastic culturing effect to the container surface that has not been seen before.

[0024]

The present invention will be described below in more detail with reference to examples. Embodiment 1 FIG. 2 g of maltopentaose was dissolved in 15 ml of distilled pyridine, and this was added to distilled acetic anhydride at 4 ° C.
ml was slowly added dropwise. After stirring at 4 ° C. for 12 hours, the mixture was replaced with chloroform and washed with a dilute aqueous sodium hydrogen carbonate solution. Recrystallization from 2-propanol was repeated twice to obtain 2.58 g of acetylated maltopentaose. The yield in this case was 69%.

The reaction was confirmed by IR spectrum (NaC
In l), it was confirmed that the peak around 3500 cm -1 based on the hydroxyl group disappeared and the peak based on the ester bond appeared at 1750 cm -1. Proton NM
In R (solvent, CDCl ₃ ), an acetyl group (1.8)
-2.3 ppm) and the theoretical value of the integral value of the proton derived from the sugar skeleton (3.4-5.7 ppm) of 1.46, the experimental value was 1.50. confirmed. At this time, two spots were recognized on silica gel thin layer chromatography (hereinafter, referred to as TLC) (Rf = 0.40, 0.15; solvent, toluene: ethyl acetate = 1: 4 volume ratio). This is because the carbon at the anomeric position is optically active and separated by the difference in polarity between the α-form and the β-form. Here, the following reaction was carried out without separating the two.

Acetylated maltopentaose 2.0 g
And 0.78 g of equimolar 1,2-octadecylglycerol were added to the powder 1 of Molecular Receive 4A.
After dissolving in 45 ml of dichloroethane in the presence of 2 g, 1.5 ml of MSCF was added dropwise and stirred at room temperature for 65 hours.
Further, after filtration and concentration, purification was performed using a silica gel column (solvent, toluene: ethyl acetate = 1: 1 by volume) to obtain an acetylated product of the oligosaccharide derivative of the present invention.

The acetylated product of the oligosaccharide derivative of the present invention obtained here showed two spots on TLC (Rf = 0.43, 0.22; solvent: toluene: ethyl acetate = 3: 1). Capacity ratio). Here, those having a high Rf value were the α-form and 150 mg, and those having the low Rf value were the β-form and 460 mg, and the total yield was 23%. To confirm this reaction, an IR spectrum and proton NMR (solvent, CDCl ₃ ) were used.
A peak based on a long chain appeared at 900 cm-1. further,
In the latter, 1.48 was obtained as an experimental value with respect to a theoretical value of 1.46 of the integral ratio of protons derived from the alkyl group (0.7 to 1.7 ppm) and the acetyl group (1.9 to 2.3 ppm),
From each result, it was confirmed that the expected reaction was performed.

The deacetylation of the acetylated product of the oligosaccharide derivative of the present invention was carried out by dissolving the α-form or the β-form in methanol, adding a small amount of triethylamine and pure water and refluxing for 12 hours. . This was cooled to 0 ° C. and recrystallized to recover the deacetylated oligosaccharide derivative of the present invention in a yield of 69%. And, in the IR spectrum, 3500 cm -1
A peak based on a hydroxyl group appears in the vicinity, and 1750
The disappearance of the carbonyl peak near cm-1 confirmed the completion of the deacetylation reaction. Further, from the proton NMR spectrum, it can be seen that -CH ₃ (δ: 0.7 to
1.0),-(CH ₂ ) _16- (δ: 1.1 to 1.2)
And a sugar skeleton, glycerol, —OCH ₂ CH ₂ (δ:
(2.8 to 5.6) (where δ is the chemical shift (ppm)) and the theoretical value of the integral ratio of the proton is 1.17, whereas 1.13 was obtained as the experimental value. It was confirmed that glycerol was introduced into the sugar chain at a ratio of 1: 1.

The TCL (volume ratio of solvent, chloroform: methanol = 2: 1) of the oligosaccharide derivative of the present invention obtained in α-form and β-form is a monospot, and Rf of α-form and β-form is obtained. The values were 0.10 and 0.16, respectively. Finally, the carbon NM of the derivative
In R (solvent, dimethyl sulfoxide), the chemical shift of the terminal anomeric carbon is 92 pp of maltopentaose.
The shift from m to 103 ppm confirmed that dialkylglycerol was selectively introduced at the terminal anomeric position. Therefore, the oligosaccharide derivative of the present invention in this embodiment can be maltopentaose distearyl glycerol (hereinafter, referred to as MPDSG) having a clear structure.

The evaluation of the vesicle stabilization of the oligosaccharide derivative of the present invention, ie, MPDSG, in this example was performed as follows. MPDSG (3 mg) and dipalmitoyl phosphatidylcholine (hereinafter, DPPC) as a phospholipid
That. ) 100 mg was dissolved in methanol and mixed, and then a thin film was formed on the inner wall of the pear-shaped flask using a rotary evaporator. 5 ml of pure water and several glass beads were added thereto, and the mixture was stirred by heating at 50 ° C. with a vortex to obtain a cloudy liquid. This cloudy liquid was treated with an extruder, and the pore size of the polycarbonate filter at the time of treatment was set to 0.4 μm, 0.2 μm, 0.1 μm,
By passing through the filter five times while sequentially changing the particle size to 0.05 μm, a white transparent DPPC vesicle dispersion liquid was obtained. The average particle size of the obtained endoplasmic reticulum was 57 ± 12 nm and 55 ± 10 nm, respectively, as measured by a particle size distribution analyzer and a negatively stained transmission electron microscope.

After diluting the vesicle dispersion liquid to 0.5 wt%, gel filtration (Sepharose, manufactured by Pharmacia)
CL-4B, 20 mmd x 300 mmh), MPDSG not introduced into the endoplasmic reticulum was removed out of the system, and the sugar in each fraction was quantified by the phenol / sulfuric acid method, whereby the introduction rate of MPDSG into the endoplasmic reticulum was determined. It was determined. As a result, 99% of the added amount of MPDSG was incorporated in the endoplasmic reticulum. The dispersion stability of the endoplasmic reticulum passed through a 0.05 μm filter was evaluated by measuring the change over time in absorbance (turbidity) at 500 nm with a UV-visible spectrophotometer at room temperature.

FIG. 1 is a time-dependent change in turbidity (500 nm) when each of the MPDSG-modified vesicle, the sugar ester-modified vesicle, and the unmodified DPPC vesicle was used as a 0.5 wt% dispersion and left at room temperature. . As is clear from FIG. 1, in the case of the unmodified DPPC vesicle, when the sample immediately after preparation was left at room temperature, the turbidity of the solution immediately began to increase, and aggregation of the vesicle occurred. In the sugar ester-modified vesicles, the increase in turbidity reached about 70% of that in the unmodified system, and aggregation was not sufficiently suppressed. On the other hand, in the system modified by introducing the oligosaccharide derivative of the present invention, that is, MPDSG, in this example, the turbidity was increased by about 7% immediately after the preparation and standing, as compared with the unmodified system. Was hardly increased, and aggregation was suppressed. Therefore, the effect of imparting dispersion stability to the endoplasmic reticulum of the MPDSG of the present invention was extremely large.

Embodiment 2 FIG. An acetylated laminary pentaose obtained from 2 g of laminary pentaose by the same acetylation method as in Example 1 and an equimolar stearyl alcohol were also purified by the method of Example 1 to obtain an acetylated lamina. 1.5 g of leapentaose monostearyl ether was obtained (total yield, 40%). In the IR spectrum, a peak based on a long-chain alkyl group appeared at 2700 to 2900 cm -1. In proton NMR (solvent, chloroform), an alkyl group (0.7 to 1.7 pp) was used.
m) and an acetyl group (1.9 to 2.3 ppm), an experimental value of 0.80 was obtained with respect to a theoretical value of 0.73 for the proton integral ratio, confirming the progress of the ether reaction. After that, the product was deacetylated and recrystallized to recover the oligosaccharide derivative of the present invention, laminary pentaose monostearyl ether (hereinafter referred to as LPMS), in a yield of 80%.

Incidentally, the LPMS of the present invention is
From the spectrum, it was confirmed that the deacetylation reaction was completed. From the proton NMR spectrum, the integral ratio of the protons was -CH ₃ ,-(CH ₂ ) _16- , and the sugar skeleton,
In contrast to -OCH ₂ CH ₂ , the theoretical value is 1.51,
From an experimental value of 1.46, it was confirmed that stearyl alcohol was introduced at a ratio of 1: 1 to the sugar chain.

For the evaluation of the stabilization of the endoplasmic reticulum of the LPMS of the present invention, a mixed lipid powder obtained by freeze-drying containing LPMS (dimyristoylphosphatidylcholine:
Cholesterol: myristic acid: LPMS = 7: 7:
100 ml of physiological saline was added to 10 g of a (molar ratio of 2: 0.3) and stirred at 4 ° C. for 24 hours to obtain a dispersion. Using a microfluidizer at 4500 psi for 10 minutes
The mixture was treated at 4 ° C. to obtain a vesicle dispersion having an average particle size of 300 ± 130 nm. Then, the sugar content of the supernatant obtained by centrifugation was determined, and the introduction rate of LPMS was calculated. The result was 85%. After redispersion in physiological saline, dextran (molecular weight: 40,000), which is a regulator of oncotic pressure, was added.
Its stability was observed from turbidity measurements at 0 nm. In the unmodified vesicle, the turbidity increased even with the addition of 1 wt%, and strong aggregation was observed. However, in the vesicle modified with LPMS, the turbidity did not increase, indicating a strong inhibitory effect on the aggregation action of the dextran. Admitted.

Embodiment 3 FIG. Mixed lipid powder containing MPDSG of the present invention (DPPC: cholesterol: distearylphosphatidic acid: MPDSG = 10: 9: 0.9:
500 mg) were obtained by freeze-drying from benzene. 10 ml of physiological saline was added thereto, and the mixture was stirred at 4 ° C. for 12 hours, and finally passed through a filter having a pore size of 0.2 μm by an extruder to obtain a vesicle dispersion having an average particle size of 200 nm. . MPD to this endoplasmic reticulum
The introduction rate of SG was 98%. Then, in order to determine the evaluation of the stability of the endoplasmic reticulum by measuring the viscosity, physiological saline was added to the endoplasmic reticulum and redispersed with a vortex mixer to obtain a measurement sample. After adding 5 mM calcium chloride (as calcium ion) to this sample, the viscosity of the dispersion was measured by a rotational viscometer (Vismetholone VS-AK). As a result, there was no increase in viscosity and the viscosity remained at 5 cp. Therefore, the MPDSG of the present invention almost completely suppressed aggregation even when calcium ions were added to the vesicle dispersion liquid system. On the other hand, the viscosity of the unmodified vesicle dispersion liquid excluding MPDSG was markedly increased by adding calcium ions, indicating that the aggregation proceeded.

Embodiment 4 FIG. Mixed lipid powder containing MPDSG of the present invention (molar ratio of hydrogenated lecithin: cholesterol: palmitic acid: MPDSG = 7: 7: 2: 0.4) 1
0.0 g was dispersed in a purified stromal-removed hemoglobin solution (40 wt%, 200 ml), stirred at 4 ° C. for 2 hours, and then treated with a microfluidizer.
An endoplasmic reticulum dispersion of 00 ± 98 nm was obtained. Further, the particles were passed through a filter having a pore size of 0.2 μm by a particle size extrusion method, and the outer aqueous phase hemoglobin and unintroduced MPDSG were removed by gel filtration using physiological saline as a solvent, and hemoglobin having an average particle size of 203 ± 50 nm was encapsulated. A dispersion of vesicles was obtained.

The hemoglobin-containing vesicle dispersion was adjusted to pH
After adjusting to 7.4, red blood cells of human blood were added thereto to a concentration of 0.5 wt%, incubated at 37 ° C. for 2 hours, and then centrifuged at 5000 G for 30 minutes to determine the amount of hemoglobin in the supernatant. , The hemolytic properties of the endoplasmic reticulum were observed. As a result, 90% hemolysis was observed in the system in which the MPDSG of the present invention was not introduced into the endoplasmic reticulum, but only about 5% hemolysis was observed in the system into which the MPDSG of the present invention was introduced. It was confirmed.

Next, the dispersion of hemoglobin-containing vesicles was treated with a lipid concentration of 5 wt%, a hemoglobin concentration of 10 wt%, and a pH of 5 wt%.
Adjusted to 7.4, 4000 mg / kg was administered to a group of rats via the tail vein. After blood was collected over time, red blood cells and hemoglobin-containing vesicles were separated by centrifugation, and the time-dependent change in the amount of hemoglobin-containing vesicles removed from the blood was observed. As a result, the average of the blood half-lives of the system in which the endoplasmic reticulum was modified with the oligosaccharide derivative MPDSG of the present invention and the unmodified system were 38 hours and 24 hours, respectively.
It was found that the endoplasmic reticulum modification of MPDSG was effective.

[0040]

The oligosaccharide derivative of the present invention is
By using this as a modifier for phospholipid aggregates, an extremely large effect on the dispersion stability of phospholipid vesicles is recognized, and a strong phagocytosis-inhibiting effect in vivo can be expected. Further, as compared with conventional oligosaccharide fatty acid esters, both of the efficiency of synthesis and the effect of modification are excellent, which greatly contributes to the industry.

[Brief description of the drawings]

FIG. 1 is a time-dependent change in turbidity (500 nm) when a DPPC vesicle (0.5 wt%) using an oligosaccharide derivative according to the present invention is left at room temperature.

[Explanation of symbols]

The numbers on the vertical axis represent turbidity (0.D.), and the numbers on the horizontal axis represent retention time (day). In addition, ● represents an unmodified DPPC vesicle, ○ represents an MPDSG vesicle, and Δ represents a sugar ester-modified vesicle.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI B01F 17/56 B01F 17/56 B01J 13/02 C07H 15/10 C07H 15/10 C08B 37/00 J C08B 37/00 B01J 13/02 Z ( 72) Inventor Hiromi Sakai 1-6-9, Sekicho Minami, Nerima-ku, Tokyo (72) Inventor Hidetoshi Tsuchida 1-10-10, Sekichominami, Nerima-ku, Tokyo (56) References JP-A-4-89494 ( JP, A) JP-A-5-255369 (JP, A) "The 43rd Meeting of the Symposium on Colloid and Interface Science" Published by The Japan Chemical Society, Colloid and Surface Chemistry Subcommittee (September 21, 1990) ), “1B08 main” column (58) Fields investigated (Int. Cl. ⁷ , DB name) C07H 15/04 C07H 15/10 A61K 9/127 A61K 47/26 A61K 47/36 B01F 17/56 B01J 13 / 02 C08B 37/00 CAPLUS (STN)

Claims (7)

(57) [Claims] An oligosaccharide derivative having a sugar polymerization degree represented by an integer of 3 to 20 and a hydrophobic group introduced at an anomeric position of a reducing terminal group through an ether bond, wherein the hydrophobic group is (1) A) a straight-chain alkyl group having 12 to 22 carbon atoms; (2) a dialkylglycerol group in which an alkyl group having 12 to 22 carbon atoms is bonded to the 1 and 2 positions of glycerin; and (3) an alkenyl chain having 12 to 22 carbon atoms. Oligosaccharide derivatives that are: 2. A phospholipid vesicle dispersion stabilizer comprising the oligosaccharide derivative according to claim 1 . 3. The phospholipid vesicle dispersion stabilizer according to claim 2, wherein the hydrophobic group of the oligosaccharide derivative is an alkyl chain having 12 to 22 carbon atoms. 4. The phospholipid vesicle dispersion stabilizer according to claim 2, wherein the hydrophobic group of the oligosaccharide derivative is an alkenyl chain having 12 to 22 carbon atoms. 5. A phospholipid according to claim 1 oligosaccharide derivative er for producing phospholipid compositions containing 0.01 to 20 mol% relative to the lipid according. 6. The phospholipid composition for producing endoplasmic reticulum according to claim 5, wherein the hydrophobic group of the oligosaccharide derivative is an alkyl chain having 12 to 22 carbon atoms. 7. The phospholipid composition for producing an endoplasmic reticulum according to claim 5, wherein the hydrophobic group of the oligosaccharide derivative is an alkenyl chain having 12 to 22 carbon atoms.