JPH0455433B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a polymerizable glycerophospholipid. [Prior art] Natural phospholipids are mainly composed of glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, and sphingophospholipids such as sphingomyelin, and are used to form biological membranes of cells. It survives as an important component of Among these, phosphatidylcholine is generally called lecithin and is the most abundant phospholipid in the animal and plant kingdoms. A common feature of these phospholipids is that they have a hydrophilic group and a hydrophobic group, and they form a double layer in biological membranes, and together with proteins and cholesterol, they are essential for biological phenomena such as permeation of substances and selective transport. It performs functions that cannot be performed. Phospholipids are already used industrially as natural emulsifiers, and are used in the food industry, cosmetics industry, etc. Recently, it has become known that phospholipids form very small closed endoplasmic reticulum called liposomes in aqueous solutions due to their lipid characteristics. Many attempts have been made to fill these liposomes with drugs and use them as a drug delivery system. However, because liposomes currently use natural phospholipids, mainly lecithin, as raw materials, they are easily attacked by enzymes in the living body, and when liposomes containing a drug are injected into a living body, the drug is transported locally. The drawback is that the liposomes are destroyed over time, and the drug's efficacy is not fully demonstrated. Various attempts have been made to compensate for these drawbacks. One type of phospholipid is one in which the phospholipid itself is resistant to enzymes, such as an alkyl-type phospholipid that originally has an ester bond structure but has been converted to an ether bond structure. be. The other type has an ester bond structure, but it also has a polymerizable group. In the latter case, the property of phospholipids being regularly oriented in an aqueous solution is utilized, and by polymerization, liposomes that are resistant to enzymes and also resistant to pressure from the outside world can be obtained.
As such polymerizable phospholipids, those having a methacrylic acid group (Japanese Unexamined Patent Publication No. 152816-1982) and those having an acetylene group (Japanese Unexamined Patent Publication No. 135492-1982) have been proposed. [Problems to be solved by the invention] However, because such conventional polymerizable phospholipids introduce groups that are not derived from natural products, they are difficult to be used by living organisms as foods, cosmetics, medicines, etc., and are susceptible to pressure from the outside world. In addition to being vulnerable to
There was a problem that drug release and metabolism were not carried out effectively. The present invention is intended to solve these conventional problems.Since it is bioavailable and has appropriate resistance to enzymes, it can be used in drug delivery systems, and can also be used in drug delivery systems. The purpose of the present invention is to provide a novel polymerizable glycerophospholipid that exhibits resistance to. [Means for Solving the Problems] The present invention is a polymerizable glycerophospholipid comprising a compound represented by the following general formula [I]. (In the formula, R ¹ CO- and R ² CO- each represent a naturally occurring fatty acid residue, one or both of which is a conjugated fatty acid residue having a conjugated double bond, and R ³ is -CH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ , −CH ₂ CH ₂ N ⁺
_H3 ,

[Formula]H,

【formula】

[expression] or

[Effect]

The glycerophospholipid of the present invention has polymerizability and is used for producing a glycerophospholipid polymer. Since this glycerophospholipid forms liposomes in an aqueous solution and is regularly oriented, polymerization in this state produces a uniform thin film-like polymer. The resistance of this polymer is that its decomposition rate is 10 to 100, when the degradability of lecithin to enzymes is 100.
30, so when used in a drug delivery system, it will not be broken down by enzymes until it reaches the affected area, and once it reaches the affected area, it will be broken down, effectively releasing the drug inside.
The polymer itself is metabolized and does not accumulate, and the result is a polymer that is resistant to external pressure. The polymerization can be carried out in the same manner as the conventional polymerization of polymerizable phospholipids, and the polymerization rate is high. In the case of liposomes, a method of polymerizing in an aqueous solution by irradiation with ultraviolet rays is suitable. Depending on the purpose, polymerization can also be carried out using water-soluble or oil-soluble peroxides. The glycerophospholipid or polymer thereof of the present invention can be used in living organisms as foods, cosmetics, medicines, etc. [Effects of the Invention] According to the present invention, a conjugated fatty acid residue, which is a naturally occurring fatty acid residue and at least one of which has a conjugated double bond, is introduced, so that it has no inhibitory effect even when used in living organisms. The result is a polymerizable glycerophospholipid that has moderate resistance to enzymes and high resistance to pressure from the outside world. Furthermore, since this glycerophospholipid has excellent polymerizability, it can be used as a polymerized liposome in a drug delivery system. In this case, polymerized liposomes also have appropriate resistance to enzymes, so they can efficiently transport the drug to the affected area and then release it effectively, and the polymerized liposome itself can also be metabolized. No accumulation, no destruction or aggregation due to temperature changes,
It exhibits high resistance to external pressure, with little leakage of contents. [Example] The present invention will be explained below using reference examples and examples. Reference example 1 100g of tung oil was dissolved in methanol.
24g of KOH and methanol at reflux temperature
Time saponified. The temperature was maintained at 50 to 60°C, the pH was lowered to 1 to 2 with an aqueous hydrochloric acid solution, and the liberated fatty acids were extracted with hexane to obtain mixed fatty acids. The fatty acid composition of this fatty acid is 83% eleostearic acid, 8% linoleic acid, 7% oleic acid, and 1% stearic acid.
%, palmitic acid 1%. This mixed fatty acid hexane solution was washed with water and then dehydrated with anhydrous sodium sulfate. This solution was recrystallized in a refrigerator at 5°C. The crystals were filtered off, dissolved in hexane again, and recrystallized. After filtering the crystals, dry them at room temperature under reduced pressure.
41 g of white scaly crystal fatty acid was obtained. The eleostearic acid in this fatty acid was 99%. Reference Example 2 50 g of commercially available egg yolk lecithin was separated using 1.5 silica gel packed in a glass column with a diameter of 80 cm.
The elution solvent is chloroform/methanol/water =
A mixed solvent of 65/25/4 was used. Each fraction was tracked by thin film chromatography, and 31 g of lecithin was obtained from the lecithin portion, and 9 g of phosphatidylethanolamine was obtained from the phosphatidylethanolamine portion. 500ml of the lecithin obtained
Tetrabutylammonium hydroxide 10% methanol solution is dissolved in ether and stirred.
Added 80ml. Glycerylphosphorylcholine precipitated at the bottom was recovered by tectonation and washed with ether. The amount of glycerylphosphorylcholine obtained was 9.1g. This glycerylphosphorylcholine was dissolved in warm water and mixed with 18.9 g of cadmium chloride 2.5 hydrate, which had been separately dissolved in warm water, and reacted. Insoluble matter was filtered out, and ethanol was added to this glycerylphosphorylcholine-cadmium chloride complex aqueous solution until it became cloudy.
The mixture was left at ℃ overnight, and the resulting crystals were collected. These crystals were again dissolved in warm water, and ethanol was added thereto for crystallization. Collect and dry the crystals,
11.8 g of glycerylphosphorylcholine-cadmium chloride complex was obtained. Example 1 Eleostearic acid obtained in Reference Example-1
16.8g was dissolved in dry chloroform, 6.2g of dicyclohexylcarbodiimide was added, and the mixture was reacted overnight at 5°C to synthesize eleostearic anhydride.
The by-produced dicyclohexyl urea was filtered off, and 4.4 g of the glycerylphosphorylcholine-cadmium chloride complex of Reference Example 2 and 2.5 g of dimethylaminopyridine were added to the chloroform solution of eleostearic anhydride. The reaction was allowed to proceed for 48 hours with stirring at room temperature, and after the reaction, the precipitate was filtered and chloroform was removed to obtain crude lecithin. The crude lecithin was redissolved in a methanol/chloroform mixed solvent, 10 g each of ion exchange resins Amberlite IRC-50 and IRA-45 (trademarks of Rohm and Haas) were added, and residual cadmium chloride was removed in batches. Crude lecithin was obtained again by removing methanol. This crude lecithin was subjected to column fractionation using a 5 cm diameter column packed with 0.5 silica gel using a mixed solvent of chloroform/methanol/water = 65/25/4 as an eluent. Collect the lecithin part while tracking with TLC,
After desolvation, pure 1,2-diereostearoyl-
5.4 g of Sn-3-glycerophosphorylcholine was obtained. Obtained 1,2-diereostearoyl-Sn
The elemental analysis values of -3-glycerophosphorylcholine are as follows. Elemental analysis value C H N (wt%) Theoretical value 67.95 9.78 1.80 Actual value 66.81 9.46 1.81 Also, the above 1,2-diereostearoyl-Sn
The NMR spectrum of -3-glycerophosphorylcholine is as shown in FIG. 1, and the position codes added to FIG. 1 are as shown in the formula [ ]. 200 mg of 1,2-diereostearoyl-Sn-3-glycerophosphorylcholine obtained above
was dissolved in benzene, and debenzened in a 50 ml eggplant flask under reduced pressure using a rotary evaporator to adhere a film of 1,2-diereostearoyl-Sn-3-glycerophosphorylcholine to the flask wall. 12.5 ml of ion-exchanged water was added to the flask, and liposomes were formed using a probe-type ultrasonic generator. After ultrasonication at 35 watts for 20 minutes, 5 ml of the solution was taken and placed in a 1 cm quartz cell. The quartz cell was kept at 30°C and degassed for 2 hours while bubbling nitrogen. The quartz cell containing this degassed liposome solution was irradiated with ultraviolet rays for 3 hours to cause polymerization to form polymerized liposomes. When this liposome was observed under an electron microscope, it was found to be a unilamellar liposome with a diameter of about 300 mm. Furthermore, polymerized phosphatidylcholine was obtained by adding benzene to this liposome aqueous solution and performing azeotropic dehydration. As a result of IR measurement, absorption due to conjugated double bonds around 1600 cm ^-1 had disappeared, and the average molecular weight was approximately 38000. Example 2 30 g of eleostearic acid (cis-9, trans-11, trans-13) obtained in Reference Example-1 was dissolved in 300 ml of hexane, and 0.3 g of iodine was added to isomerize at room temperature to obtain all-trans form. Eleostearic acid was obtained. The product was then purified by recrystallization in the same manner as in Reference Example-1. This all-trans eleostearic acid (trans-9, trans-11, trans-13)
16.8g was reacted and purified in the same manner as in Example-1 to obtain all-trans 1,2-diereostearoyl-Sn-3.
- 5.2 g of glycerophosphorylcholine was obtained. The elemental analysis values of the obtained all-trans 1,2-diereostearoyl-Sn-3-glycerophosphorylcholine are as follows. Elemental analysis value C H N (wt%) Theoretical value 67.95 9.78 1.80 Actual value 67.22 9.39 1.80 200 mg of all-trans 1,2-diereostearoyl-Sn-3-glycerophosphorylcholine obtained above was dissolved in chloroform, Chloroform was removed under reduced pressure using a rotary evaporator in a test tube, and all-trans 1,2-diereostearoyl-Sn-3-glycerophosphorylcholine was adhered to the test tube wall in the form of a film. 12.5 ml of ion-exchanged water was added to the test tube and stirred for 1 hour using a test tube mixer to form liposomes. Put 5 ml of liposome solution into a 1 cm quartz cell, Example-
Polymerized liposomes were formed in the same manner as in 1. This liposome was observed with an electron microscope and had a diameter of 500 mm.
~3000 angstrom liposomes.
Polymerized phosphatidylcholine was obtained by adding benzene to this polymerized liposome aqueous solution and performing azeotropic dehydration. As a result of measuring with IR, 1600~1650cm ^-
The absorption due to the conjugated double bond near ¹ has disappeared,
The average molecular weight was 82,000. Example 3 2.8 g of parinaric acid was dissolved in dry chloroform, 1.1 g of dicyclohexylcarbodiimide was added, and the mixture was reacted overnight at 5° C. to synthesize parinaric anhydride. The by-produced dicyclohexyl urea was filtered out, and glycerylphosphorylcholine of Reference Example-2 was obtained.
Add 0.55 g of cadmium chloride complex and 0.4 g of dimethylaminopyridine to this parinaric anhydride chloroform solution, react and purify in the same manner as in Example-1 to obtain 1,2-diparinaloyl-Sn-3.
-0.71 g of glycerophosphorylcholine was obtained. Obtained 1,2-diparinaroyl-Sn-3-
The elemental analysis values of glycerophosphorylcholine are as follows. Elemental analysis value C H N (wt%) Theoretical value 68.31 9.31 1.81 Actual value 68.15 9.22 1.79 Test example 1 (freeze-thaw test) The polymerized liposome sample obtained in Example 1 was frozen (liquid nitrogen) and thawed (room temperature). 10 operations
The freeze-thaw test was repeated several times. When the state of the solution was observed after the test, it remained a transparent liposome dispersion. As a comparative example, polymerized liposomes were formed according to Example 1 using polymerizable glycerophospholipids of compounds [1] to [4] below. A freeze-thaw test was conducted using this polymerized liposome in the same manner as above. The results are shown in Table 1. (Here, n=8, m=1, l=17.)

[Table] As is clear from Table 1, the polymerized liposome of Example 1 was prepared using conventional polymerizable glycerophospholipids [1] to
It can be seen that it is more stable against temperature changes than the polymerized liposome made of [4]. It is presumed that the reason why precipitates are generated in the polymerized liposomes composed of compounds [1] to [4] is that the polymerized liposomes in the sample are destroyed by temperature changes. Test Example 2 (Leakage Test) For each of the polymerizable glycerophospholipid obtained in Example 1 and the polymerizable glycerophospholipids of compounds [1] to [4] shown in Test Example 1, 5 (6 )-A polymerized liposome dispersion containing carboxyfluorescein (CF) was prepared. That is, after each phospholipid (0.2 g) was dissolved in chloroform, the chloroform was slowly removed under reduced pressure using a rotary evaporator so as to form a thin film on the flask wall, and the solution was vacuum-dried overnight. Next, transfer to the flask with the phospholipid thin film attached.
Inject phosphoric acid shock solution (PH7.0, 20 ml) containing CF at a concentration such that 100 mM CF is encapsulated in the liposome aqueous phase, and after sufficient hydration, ultrasonic irradiation (Tomy Seiko Co., Ltd. UR-200P) was applied. , trademark, output 30W) for 15 minutes under ice cooling to prepare a liposome dispersion. These liposome dispersions were subjected to γ-ray irradiation polymerization (0.34 Mrad) at 25°C, then purified using a gel column (Sepharose CL-4B), and used as samples for leakage tests. In the CF leakage test, these dispersions were heated to 50°C to promote CF leakage, and after 2 hours, each dispersion was separated from the CF leaked from the polymerized liposomes using a gel column, and the fluorescence intensity was measured using a spectrometer ( The leakage rate (%) was determined by quantitative determination using HPF-4 (trademark, manufactured by Hitachi, Ltd.). The results are shown in Table 2.

[Table] As is clear from Table 2, the polymerized liposome of Example 1 was prepared using conventional polymerizable glycerophospholipids [1] to
It can be seen that it has higher resistance to external pressure than the polymerized liposome made of [4]. Test Example 3 (Enzyme Resistance Test) Polymerizable glycerophospholipids obtained in Examples 1 and 2, egg yolk lecithin (EYPC) as a comparative example, and compounds [1] and [3] shown in Test Example 1 above. ,
Resistance to enzymes (phospholipase A2, phospholipase D) was measured for the polymerizable glycerophospholipid [4]. Table 3 shows the values when the decomposition rate of egg yolk lecithin is set as 100.

[Table] As is clear from Table 3, the enzyme resistance of the polymerizable glycerophospholipids of Examples 1 and 2 is higher than that of egg yolk lecithin, and glycerophospholipids [1] and [ 3], [4]
It can be seen that it is lower than

[Brief explanation of the drawing]

FIG. 1 is an NMR spectrum diagram in Example-1.

Claims (1)

[Scope of Claims] 1. A polymerizable glycerophospholipid comprising a compound represented by the following general formula [I]. (In the formula, R ¹ CO- and R ² CO- each represent a naturally occurring fatty acid residue, one or both of which is a conjugated fatty acid residue having a conjugated double bond, and R ³ is -CH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ , −CH ₂ CH ₂ N ⁺
H ₃ , [formula]H, [formula] [formula] or [formula]). 2. The polymerizable glycerophospholipid according to claim 1, wherein R ¹ CO- is an eleostearic acid residue or a parinaric acid residue. 3. The polymerizable glycerophospholipid according to claim 1 or 2, wherein ^R2CO- is an eleostearic acid residue or a parinaric acid residue. ^4. The polymerizable glycerophospholipid according to claim ₂ or 3, wherein R3 is _-CH2CH2N ⁺ ( _CH3 ) ₃ .