JP3130135B2 - Stabilizer for phospholipid endoplasmic reticulum - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilizer for vesicles comprising a disaccharide derivative having a well-defined structure, which is useful for imparting stability to phospholipid vesicles (hereinafter referred to as "vesicles"). . That is, by using the stabilizer for vesicles of the present invention as a modifier for vesicles, in addition to suppressing aggregation of vesicles themselves, stabilizing dispersion against ions, or reducing the residence time of vesicles in vivo. Can be extended.

[0002]

2. Description of the Related Art As is well known, vesicles usually have instability such as aggregation and fusion, morphological changes and leakage of inclusions accompanying the above-mentioned phenomena. Many proposals have been made. First, a solution by polymerization of an aggregate of phospholipids having an unsaturated group has been attempted (for example, review: HR).
ingsdof et al. Angew. Che
m. , Int. Engl. , 27, 113 (198
8)), the membrane polymerization loses the properties of the phospholipid, for example, gel-liquid crystal phase transition, phase separation, fluidity of the membrane, and the like, resulting in loss of functions related thereto.

On the other hand, a cholesterol derivative having β-aminogalactose has been used for controlling liposome aggregation (PS Wu et al., Proc. N).
atl Acal. Sic. , USA, 78, 6211.
(1981)), however, the effect of the derivative is not as great as expected, suggesting that the degree of polymerization of the sugar is strongly involved in the effect. In addition, a hyaluronic acid derivative having a liposome emulsifying action and an emulsifying system stabilizing action has been disclosed (JP-A-3-47801). However, in the hyaluronic acid derivative, since the acyl group is non-selectively ester-bonded, the stabilizing effect cannot be effectively obtained, and the effect largely depends on the introduction position and the number of the acyl groups. . In addition, the viscosity sometimes becomes high and may not be practical.

[0004] The present inventors have already introduced the oligosaccharide fatty acid ester developed by the present inventors into the endoplasmic reticulum bilayer membrane, thereby inhibiting the aggregation and fusion of the endoplasmic reticulum, ie, dispersing the endoplasmic reticulum. It discloses that it is extremely effective for stabilization (Japanese Patent Laid-Open No. 1-294701).
In that case, the ester derivative of oligosaccharide having a long sugar chain shows a better effect, and when the disaccharide is used as a base, a sufficient effect is not obtained. JP-A 1-2947
Although the invention disclosed in Japanese Patent Publication No. 01-2001 brings a number of solutions to the stability of the endoplasmic reticulum, reactive groups (hydroxyl groups)
The introduction of a long-chain fatty acid group into an oligosaccharide having a large number of is carried out non-selectively, and the number and site of introduction are various.

[0005]

The oligosaccharide fatty acid ester developed by the present inventors is, as described above,
Since the introduction of long-chain fatty acid groups is non-selective and the number and site of introduction are various, separation and purification are complicated and the yield is low. There is a problem that the difficulty of being fixed on the surface differs depending on the isomer of the oligosaccharide. 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, the development of a stabilizer for phospholipid vesicles comprising a disaccharide derivative having a well-defined structure in which a hydrophobic group is selectively introduced into a specific group of a disaccharide has been technically proposed. Make it an issue.

[0006]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the present invention provides a disaccharide having a structure and effect clearly obtained by selectively introducing a hydrophobic group at the anomeric position of a disaccharide. The present invention relates to providing an ER stabilizer comprising a derivative. That is, the present invention relates to a stabilizing agent for an endoplasmic reticulum comprising a disaccharide derivative in which a reducing end group of a disaccharide is formed of aldose, and a hydrophobic group is introduced at an anomeric position of the reducing end group by an amide bond. Things.

Hereinafter, the present invention will be described in detail. The disaccharide, which is the base of the disaccharide derivative used for the stabilizer for the endoplasmic reticulum of the present invention, is a disaccharide having a reducing terminal group composed of aldose.

In the disaccharide derivative used for the stabilizer for the endoplasmic reticulum of the present invention, since the balance between hydrophilicity and hydrophobicity needs to be emphasized, the hydrophobic group having a hydroxyl group used herein is, for example, an alkyl group having an amino group. , The carbon number is 12 ~
When it is 22 and has an unsaturated bond, the number of unsaturations is preferably from 1 to 4.

In the disaccharide derivative used as the stabilizer for the endoplasmic reticulum of the present invention, a compound having a primary amino group as a hydrophobic group at an amide bond at the anomeric position,
That is, an amine is used. Thus, if the amines that can be used in the present invention are exemplified, examples of the aliphatic primary amine include laurylamine, cetylamine, stearylamine, behenylamine, oleylamine, and the like.In phospholipids, phosphatidylethanolamine or phosphatidylserine And the like.

The introduction of a hydrophobic group into the terminal anomeric position of a sugar chain by an amide bond is carried out as follows. That is, the reducing end of the disaccharide is oxidized with hypoiodic acid, the anomeric position is opened as a carboxylate, and then neutralized by a cation exchange column to obtain a carboxylic acid. The carboxylic acid is sequentially dehydrated with ethanol and methanol to obtain a product having a reducing terminal group as a lactone ring.
Next, the product is dissolved in methanol, formamide or dimethylformamide, and then mixed with a phospholipid having an aliphatic primary amine or a primary amine dissolved in methanol or chloroform to form a homogeneous solution. The reaction is carried out at a temperature of 90 ° C, preferably 40 to 70 ° C, to obtain the desired disaccharide derivative. The obtained disaccharide derivative is purified by washing with water and hexane and using a reversed-phase and normal-phase silica gel columns.

The purity of the disaccharide derivative by the amide bond obtained by the above method can be confirmed by infrared spectrophotometer (hereinafter referred to as IR), silica gel based thin layer chromatography, or reverse phase system. And high performance liquid chromatography (hereinafter referred to as HPLC) using a normal phase silica gel column and a differential refraction detector as a detector.

The stability of vesicles to which the stabilizer for vesicles of the present invention has been applied can be confirmed as follows. Phospholipids, or cholesterol phospholipids and membrane stabilizers, cholesterol and other sterols,
Alternatively, 5 to 50 mol% of the stabilizer for the endoplasmic reticulum of the present invention is added to a mixed lipid of a phospholipid and a negatively charged lipid such as stearic acid and dicetyl phosphate, preferably 10 mol%.
A hydrate is prepared by the above addition. A vesicle dispersion having a particle size 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 obtained here can be confirmed by a particle size distribution analyzer or a transmission electron microscope negatively stained.

The dispersion of the vesicles is treated by gel filtration or ultracentrifugation to remove the stabilizer not introduced into the vesicles out of the system. Thus, the rate of introduction of the stabilizer for the endoplasmic reticulum into the endoplasmic reticulum can be determined. 90% or more of the added amount of the stabilizer for the endoplasmic reticulum of the present invention is introduced into the endoplasmic reticulum. Dispersion stability is measured with a visible spectrophotometer at 500 ° -800 nm absorbance (turbidity) of a dispersion obtained by removing the free vesicle stabilizer by gel filtration or ultracentrifugation at room temperature. By doing so, the evaluation can be confirmed.

As a method of evaluating dispersion stability,
In addition to the turbidity measurement method, for the dispersion of the endoplasmic reticulum, a method of measuring the viscosity with a cone plate type rotational viscometer,
And a microscopy method using an optical microscope or an electron microscope.

[0015]

The stabilizer for the endoplasmic reticulum of the present invention has a well-defined structure and has a very good affinity for phospholipids, so that it is suitable as a modifier for phospholipid aggregates. Furthermore, the stabilizing agent for the endoplasmic reticulum introduced into the phospholipid aggregate retains the state in which the sugar structure of the disaccharide derivative is exposed to the aqueous phase and is uniformly dispersed on the membrane of the phospholipid aggregate. .

The stabilizer for the endoplasmic reticulum of the present invention is capable of suppressing leakage of the contents of the endoplasmic reticulum, and further includes, for example, parenting, inhibition of aggregation of the endoplasmic reticulum, prevention of membrane fusion, labeling for recognition with a specific antibody, biomarker, It can be used properly according to the purpose of use such as suppression of phagocytosis in the body. In addition, the stabilizer for the endoplasmic reticulum of the present invention,
The modified vesicle dispersion has almost no increase in viscosity.

For example, when a carrier containing a functional molecule is administered to a living body through the inner aqueous phase of the endoplasmic reticulum, the longer the blood retention time, the higher the effectiveness. Aggregation by interaction with molecules and calcium ions, etc., is usually excluded from the blood in a short time by incorporation into the reticuloendothelial system and phagocytes, but the ER stabilizing agent of the present invention The residence time of the endoplasmic reticulum in blood can be controlled by surface modification of the endoplasmic reticulum.

[0018]

The present invention will be described below in more detail with reference to examples. Embodiment 1 FIG. 5.20 g of iodine was dissolved in 50 ml of a heated methanol solution at 40 ° C., and an aqueous solution of 3.50 g of maltose dissolved in 10 ml of pure water was added thereto.
For 30 minutes. Next, 125 ml of a 4% potassium hydroxide / methanol solution was added dropwise thereto over a period of 40 minutes, and it was confirmed that the iodine color had disappeared. The crystalline precipitate deposited there was filtered off with a glass filter (G4). The crystalline precipitate is washed with methanol, ethanol and ether. As a result of measurement at this stage by IR, 1650 cm ⁻¹
Since a peak derived from a potassium carboxylate salt appeared in the vicinity, it was confirmed that the anomeric position of maltose was opened as a potassium carboxylate salt.

The aqueous solution of the crystal obtained is converted into a carboxylic acid using Amberlite IR-120B, which is a cation exchange resin, and then dehydrated by adding ethanol (in this case, methanol may be used), and then maltose lactone. I got As a result of measurement at this stage by IR, 1650 cm ⁻¹
The peak around disappeared, and a peak based on the CＯO stretching vibration of the sugar skeleton was observed around 1740 cm ⁻¹ ,
It was confirmed that the maltose reducing terminal group was closed as a lactone ring. The maltose lactone obtained here is 2.
52 g, and the yield was 72.3%.

After dissolving 2.00 g of the maltose lactone in 20 ml of refluxing methanol, 50 m
By adding 1.45 g of cetylamine dissolved in 1 liter of methanol and refluxing for 3 hours, cetylamine was introduced into maltopentaose through an amide bond to obtain a crude sample of maltose cetylamide (hereinafter referred to as MCA). Was. The obtained MCA was dried under reduced pressure at 40 ° C. for 16 hours, and then impurities were extracted into a hexane layer by solid-liquid extraction using hexane. Thereafter, MCA as a residue was dissolved again with methanol, and liquid chromatography was performed. Unreacted substances were removed using a Licroprep RP-18 column (octadecyl-modified silica column only) as a stationary phase and a methanol / pure water mixed solvent as a mobile phase. As a result, 1.91 g of purified MCA was obtained. Obtained. The yield is 5
It was 6.9%.

The purified MCA was confirmed using HPLC, but was analyzed using a Capsule Pack C18 column (mobile phase: 95
% Methanol aqueous solution, flow rate: 0.5 ml / min. ) RT value = 20.67 min. Only confirmed. Further, as a result of IR measurement, a peak based on an alkyl chain was confirmed at around 2800 to 2950 cm ^-1 . Furthermore, the peak based on the C = O stretching vibration of the amide bond is 1650 cm.
^-1 and a peak based on the NH deformation angle was observed near 1550 cm ^-1 . Therefore, the obtained M
CA can be said to have a cetyl group introduced via an amide bond at the anomeric position of the reducing end group of maltose.

The modification to the endoplasmic reticulum can be carried out by the above-mentioned purified MCA3.
96 mg was dissolved in 1 ml of methanol, and 5 ml of a methanol solution in which 100 mg of dipalmitoylphosphatidylcholine (hereinafter, referred to as DPPC) was dissolved.
The mixture was used to form a thin film on the inner wall of the pear-shaped flask using a rotary evaporator.
Thereafter, 5 ml of pure water was added, and the mixture was stirred with a probe-type ultrasonic stirrer to obtain a milky white transparent MCA-modified vesicle dispersion. After the dispersion was allowed to stand at 50 ° C. for 2 hours, the particle size of the vesicles was measured by a particle size distribution analyzer, and the average particle size was 41 ± 19 nm. Similarly, MCA10
The average particle size of the mol% modified endoplasmic reticulum is 35 ± 13 nm,
The average particle size of the 20 mol% modified vesicle was 36 ± 10 nm, and values substantially unchanged even with the 15 mol% and 25 mol% modified vesicles were obtained.

The dispersion stability of the MCA-modified vesicle was determined by DPP
The addition ratio of MCA to C was set to 5 mol%, 10 mol%, 15 mol%, 20 mol%, and 25 mol%, respectively.
Immediately after the production of each modified ER and 25
The change in absorbance (turbidity) at 500 nm at 500 ° C. for 3 days was evaluated using a visible spectrophotometer. As a result, the increase in turbidity was 5 mol%> 10 mol%> 15 mol%> 20 mol% ≦ 25 mol% in terms of the MCA addition rate, and the turbidity increase rate decreased with an increase in the amount of MCA added. It was found that the stability of the endoplasmic reticulum was enhanced. A comparison of the turbidity increase immediately after the preparation of the endoplasmic reticulum and that of the endoplasmic reticulum after being left at 25 ° C. for 3 days shows that the turbidity increase rate of the unmodified DPPC endoplasmic reticulum was 100%.
When the MCA-modified ER was added, the turbidity increase rate was clearly suppressed to 15.6% when 20 mol% of MCA was added, and it was confirmed that the dispersion of the ER could be effectively stabilized by the MCA modification. Was.

The results are shown in Table 1 together with the results of Comparative Examples 1 to 5 described below. Table 1 shows a 5 wt% hydrated DPP.
It shows the turbidity increase immediately after the preparation of the C vesicle solution and after standing at 25 ° C. for 3 days. The turbidity increase rate of unmodified DPPC vesicles (referred to as increase rate in Table 1) is 100.
%, And the numerical value of the turbidity increase rate of various derivative-modified DPPC vesicles with respect to the percentage was expressed by%. In the table, the amount in parentheses indicates the amount of the modifier added, and the number indicates mol%. The value of the turbidity increase rate of the endoplasmic reticulum indicates that the smaller one has high stability, and from the table, the stabilizer for the endoplasmic reticulum of the present invention is extremely useful, and the results are as follows: The effect was almost comparable to the effect of the oligosaccharide derivative described later in Comparative Examples 1 and 2. That is, it was clearly shown that the stabilizing agent for vesicles of the present invention exhibited an extremely excellent effect by increasing the amount of addition.

Comparative Example 1. Using the same conditions as in the preparation of the MCA-modified vesicles described in Example 1, maltopentaosecetylamide (hereinafter referred to as MPCA) was introduced into the 5 wt% hydrated DPPC vesicles at a molar ratio of 5 mol%. As a result, the average particle size of the MPCA-modified DPPC endoplasmic reticulum was 32 ± 9.
nm, and the turbidity increase was 10.3% relative to the turbidity increase rate of the unmodified DPPC endoplasmic reticulum.

[0026]

[Table 1]

Comparative Example 2 As in Comparative Example 1, maltopentaose monopalmitate (hereinafter, referred to as MPMP) was introduced into 5 mol% DPPC vesicles. As a result, the average particle size of the MPMP-modified DPPC endoplasmic reticulum was 34 ± 1.
The turbidity increase was only 11.2% with respect to the turbidity increase rate of the unmodified DPPC vesicle, and the effect of stabilizing the dispersion of the vesicle was remarkable. Here, Comparative Examples 1 and 2 both use an oligosaccharide derivative as a modifier.

Comparative Example 3 As a typical example of the sucrose fatty acid ester, sugar ester (P-1695: trade name, manufactured by Mitsubishi Kasei Foods Co., Ltd.) was 5 mol%, 10 mol%,
Comparative tests were performed using 5 mol% and 20 mol% as modifiers of DPPC endoplasmic reticulum. In the case of sugar ester (P-1695) modification, the average particle size of the endoplasmic reticulum was 39 ± 14 nm when the additive rate of the modifier was 5 mol%,
At 20 mol%, it was 33 ± 7 nm and hardly changed. At 5 mol%, the turbidity increase was 65.4% with respect to the turbidity increase rate of the unmodified DPPC endoplasmic reticulum. When the amount of the modifier was increased to 20 mol%, a slight stabilization was observed at 29.1%.

Comparative Example 4 Further, when a purified product of polyethylene glycol monocetyl ether (Nonion P-208: trade name, manufactured by NOF Corporation) using commercially available polyethylene glycol having a polymerization degree of 8 was used as a modifier for DPPC vesicles, modifier 5 The average particle size of the modified endoplasmic reticulum in mole% is 4
2 ± 15 nm, and almost no change in particle size was observed even at 15 mol%. At 5 mol%, the turbidity increase was 60.2% of that of the unmodified endoplasmic reticulum.
However, at 20 mol%, the endoplasmic reticulum collapsed, and no numerical value could be obtained.

Comparative Example 5 A purified product of polyethylene glycol monostearyl ether (Nonion S-215: trade name, manufactured by NOF CORPORATION) using commercially available polyethylene glycol having a polymerization degree of 15 was also used to modify DPPC vesicles at the same molar ratio as in Comparative Example 3. A comparative test was carried out as an agent.
The average particle size of the modified endoplasmic reticulum when the modifier is 5 mol% is 3
1 ± 11 nm, and 39 ± 14 nm at 15 mol%.
And no change in particle size was observed. The increase in turbidity at 5 mol% was 39.
8%, when the amount of modifier is increased to 15 mol%,
23.0%, which was the minimum value due to the modifier. Further, when the content was 20 mol%, the collapse of the endoplasmic reticulum was observed as in Comparative Example 4, and neither the particle size nor the turbidity could be measured.

Embodiment 2 FIG. Chitobiose lactone obtained from 100.0 mg of di-N-acetyl chitobiose (hereinafter referred to as chitobiose) by the same lactonization method as in Example 1 and an equimolar oleylamine were refluxed for 3 hours under nitrogen sealing. As a result, a crude sample of G-N-acetylchitobiose oleylamide (hereinafter referred to as DACOA) was obtained. The obtained DACOA was concentrated to dryness under a nitrogen atmosphere at 40 ° C., then, impurities were extracted into a hexane layer by solid-liquid extraction using hexane, and the residue was dissolved again with methanol. The unreacted substances were removed using liquid chromatography, and purified DACOA was added to 9.3 m
g was obtained. The total yield was 9.1%.

The IR spectrum of the purified DACOA includes:
2700~3000cm around ^-1 confirmed a peak based on the alkyl chain, CH oleyl group near 700 cm ^-1
-A peak based on out-of-plane bending vibration was observed. further,
The peak based on C = O stretching vibration of the amide bond is 1650
cm- ¹ and a peak based on the NH bending angle was observed near 1550cm- ¹ . Therefore, the DACOA obtained here has an oleyl group introduced into the anomeric position of the reducing terminal group of chitobiose via an amide bond.

The mixed lipid powder containing DACOA obtained above (DPPC: cholesterol: distearylphosphatidic acid: DACOA = 10: 9: 0.9: 5 molar ratio)
250 mg were obtained by lyophilization from benzene. After adding 5 ml of physiological saline to this freeze-dried sample,
The cloudy liquid obtained by stirring for 2 hours was treated with an extruder. During the extruder treatment, the pore size of the polycarbonate filter used was set to 1.0 μm, 0.4 μm,
By passing through five times while sequentially changing to 0.2 μm, a white and transparent DACO-modified vesicle dispersion liquid (average particle size: 200 nm) was obtained. After adding 5 mM calcium chloride (as calcium ion) to the dispersion, the viscosity of the dispersion was measured with a rotational viscometer (Vismethron VS-AK) (1.2 sec ^-1 ). Was 5 cp.

On the other hand, when a vesicle dispersion is prepared in the same manner from a mixed lipid powder containing no DACOA (DPPC: cholesterol: distearylphosphatidic acid = 10: 9: 0.9 molar ratio) and calcium ions are added. Is
Since the surface of the endoplasmic reticulum was negatively charged, the endoplasmic reticulum immediately aggregated, and the viscosity increased to 11 cp.

Embodiment 3 FIG. Using cellobiose lactone obtained from 7.0 g of cellobiose by the same lactonization method as in Example 1, and equimolar distearoylphosphatidylethanolamine previously dissolved in chloroform,
This was also performed by the method of Example 1 and cellobiose distearoylphosphatidylethanolamine (hereinafter referred to as SDSP).
E. ) Was synthesized. This SDSPE was subjected to solid-liquid extraction using hexane to extract impurities into a hexane layer, and the SDSPE as a residue was dissolved again in methanol and purified by a silica gel column (mobile phase: toluene / ethyl acetate mixed solvent) to purify. 5.2 g of SDSPE was obtained, and the total yield was 24.8%. In the IR spectrum, a peak based on a stearyl group appeared at 2700 to 2900 cm ^-1 , confirming that the amide reaction had progressed.

The mixed lipid powder containing the purified SDSPE (hydrogenated lecithin: cholesterol: stearic acid:
10.0 g (SDSPE = 7: 7: 2: 4 molar ratio) was dispersed in physiological saline and stirred at 4 ° C. for 24 hours. Using a microfluidizer at 2500 psi, 1
Treated at 4 ° C. for 5 minutes, average particle size 350 ± 95 nm
Was obtained and further centrifuged (100,000 × g, 30 minutes) to obtain a lower endoplasmic reticulum layer. To see the aggregation effect of dextran, dextran (molecular weight: 40000) was added to the vesicle layer, and turbidity was measured at 800 nm. As a result, in the unmodified endoplasmic reticulum, the turbidity increased even at 1% by weight of the hydrate, and aggregation was recognized. I was able to confirm.

[0037]

EFFECTS OF THE INVENTION The stabilizer for vesicles of the present invention, when used as a modifier for phospholipid vesicles, not only inhibits aggregation of phospholipid vesicles themselves, but also Can stabilize dispersion in
The effect of suppressing phagocytosis in a living body can be obtained. Further, the use of easily available bases such as disaccharides greatly contributes to the industry.

────────────────────────────────────────────────── ─── Continued on the front page Examiner Koji Ito (56) References JP-A-1-294701 (JP, A) JP-A-5-294983 (JP, A) JP-A-5-317677 (JP, A) Table 2-502348 (JP, A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07H 15/04 C07H 15/10 C07H 9/10 CA (STN)

Claims (3)

(57) [Claims] 1. A stabilizing agent for phospholipid vesicles comprising a disaccharide derivative in which a reducing end group of a disaccharide is constituted by an aldose and a hydrophobic group is introduced by an amide bond at an anomeric position of the reducing end group. . 2. A compound having a carbon number of 12 wherein the hydrophobic group has an amino group.
A stabilizer for phospholipid vesicles comprising the disaccharide derivative according to claim 1, which has an alkyl chain of from 22 to 22. 3. The stabilization for phospholipid vesicles comprising a disaccharide derivative according to claim 1, wherein the hydrophobic group has an alkyl group having 12 to 22 carbon atoms having 1 to 4 unsaturated bonds having an amino group. Agent.