JPH0655746B2 - Polymerizable phosphatidic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel polymerizable phosphatidic acid. More specifically, the present invention relates to a polymerizable (α- or β-) capable of forming a polymeric liposome having a negative surface charge on the membrane.
-) Relates to phosphatidic acid.

[Problems to be Solved by Conventional Techniques and Inventions]

It is well known to attempt to encapsulate useful substances such as enzymes and various pharmaceuticals in microcapsules (microcapsules) and control their activity, sustained release and release.
In the initial trials, polymer compounds such as polystyrene, nylon, and cellulose acetate were used as membrane materials, and the target microcapsules were manufactured by applying the emulsification method and the interfacial polymerization method for encapsulation. Thickness is a few μm
It is thick and has a large particle size of several tens to several thousands μm. Especially, when it is used as a drug carrier and administered intravascularly, there is a problem in that it easily forms a thrombus and the toxicity of the membrane material itself. Yes, it lacked practicality.

Recently, attempts have been made to use natural or synthetic phospholipids as these membrane materials and utilize the liposomes formed by them as microcapsules. That is, film thickness 5n
m, a microcapsule consisting of a single-layer or multi-layer film with a particle size of 0.02 to several tens of μm, and a water-soluble substance in the inner aqueous phase,
A fat-soluble substance can be supported in the film. As it is a natural phospholipid, it is expected to be highly biodegradable and highly safe. Moreover, since the particle size can be controlled appropriately, thrombus formation does not occur. However, these liposomes are physically and chemically unstable in vivo, easily decomposed, and are not particularly persistent in blood. Therefore, attempts to stabilize liposomes have been earnestly made. As one of them, a method of polymerizing a bilayer membrane by using a polymerizable lipid to stabilize the membrane is known (H. Ringsdorf et al., Angevanthe Chemie International Edition English, 20).
Vol., 305 (1981) et al.). Conventionally, most of these polymerizable lipids have been phosphatidylcholine type derivatives, which are one of the biomembrane components. Phosphatidylcholine is a zwitterion and acts in the membrane as a neutrally charged lipid. Therefore, the obtained polymer liposomes (microcapsules)
Has a neutral surface charge.

However, in particular, when it is used as a drug, it is necessary to reduce the interaction with biological components (cell components, plasma proteins, cell membranes, etc.) to maintain the stability of the capsule.
Generally, under physiological conditions of pH 7.4, most of biological components are negatively charged, and if the surface charge of microcapsules is kept negative and maintained at an appropriate zeta potential, the interaction with biological components is significantly reduced. You can do it. However, such an attempt has not been carried out on a polymerized liposome because there are few examples of a polymerizable lipid having a negative charge.

In view of such circumstances, the present invention has reached the result of research to provide a material for keeping the surface charge of polymer liposomes (microcapsules) negative, that is, a novel polymerizable phospholipid having a negative charge. Is.

[Means and Actions for Solving Problems]

That is, the present invention provides (1) the general formula (However, R and R ^{1 in the} formula are (N is 6,8,10,12) an acyl group, Y is hydrogen or an alkali metal ion (Na ⁺ , K ⁺ ), and X is Represents Hereinafter, the same symbols have the same meaning. ) The polymerizable (α- and β-) phosphatidic acid represented by

Among the compounds represented by the general formula (I), the polymerizable β-phosphatidic acid derivative (general formula (III)) is synthesized, for example, as follows.

That is, 1,3-diacylglycerol represented by the general formula (IV) is mixed with a suitable anhydrous aprotic medium (methylene chloride,
After being condensed with phosphorus oxychloride in the presence of an appropriate dehydrochlorinating agent (triethylamine, trimethylamine, etc.) in chloroform, etc., the target β-phosphatidic acid (Y is Sodium ion) is obtained.

Here, the amount of the dehydrochlorinating agent is 1,3-diacylglycerol.
1.0 to 1.5 times mol, the amount of phosphorus oxychloride is 1.0 to 1.5 times mol, the reaction temperature is 0 to 30 ° C (preferably 0 to 10 ° C),
The reaction time is 3 to 24 hours (preferably 5 to 12 hours).

Compound (IV), which is an intermediate raw material, is a method for synthesizing 1,3-diacylglycerol having a saturated fatty acid chain (WG Rose, Journal of American Chemical Society, 69, 1384 (1974). Etc.)
It can be synthesized by condensation of glycerol and the corresponding fatty acid chloride (V) in the presence of a suitable dehydrochlorinating agent (quinoline or the like).

Among the compounds represented by the general formula (I), the polymerizable α-phosphatidic acid derivative (formula (VI)) is synthesized by two methods.

The first method is a polymerizable phosphatidylcholine derivative represented by the general formula (II). Is hydrolyzed using an enzyme (phospholipase D) according to a conventional method (edited by the Japanese Biochemical Society, Biochemistry Experimental Course 3, "Lipid Chemistry", page 289 (1974), etc.).

The second method is the general formula (VII) 1,2-diacylglycerol represented by the general formula (II
This is a method in which the compound is condensed with phosphorus oxychloride in the same manner as in the case of synthesizing the compound of I) and then treated with an aqueous solution of sodium hydrogen carbonate / ethylenediaminetetraacetic acid to synthesize. In addition, the compound represented by the general formula (VII), which is an intermediate raw material, is a general method for synthesizing 1,2-diacylglycerol having a saturated fatty acid chain (H. Ible, “Liposomes: From Physical Structure to Therapeutic Applications”). , Elsevier (1981), p. 24 et al.).

The production of liposomes using the polymerizable phosphatidic acid obtained as described above can be carried out according to a conventional method (G. Gregoriadis, "Liposome Technology", Volume 1, CIRP Press (1983), etc.). For example,
The polymerizable phosphatidic acid represented by the general formula (I), the polymerizable phosphatidylcholine represented by the general formula (II) and cholesterol (phospholipid / cholesterol molar ratio of 1: 2 to 2: 1) are added to dry benzene (necessarily). Depending on the solution, add a small amount of anhydrous methanol or ethanol), freeze-dry this, and add water or buffer water to the powder and heat it to room temperature to 60 ° C under an inert gas (nitrogen, argon, etc.). By vortex mixer treatment (10 to 60 minutes), multilamellar liposomes (diameter: up to several μm) are obtained. Further, by subjecting this to ultrasonic treatment (probe-type or bath-type ultrasonic transmitter), unilamellar liposomes (diameter: 20-60 nm) can be obtained. Alternatively, unilamellar liposomes can be obtained by adding water or buffer water to the powder obtained by freeze-drying and directly sonicating at room temperature under an inert gas (nitrogen, argon, etc.).
This liposome can carry various drugs, useful substances, enzymes, proteins, metalloproteins and the like.

In order to polymerize the liposomes thus obtained to produce polymer liposomes, irradiation with ultraviolet rays or gamma rays with an inert gas or a suitable initiator (azobisisobutyronitrile, azobis (2- It becomes possible by polymerizing using diaminopropane) dihydrochloride, NaHSO ₃ / K ₂ S ₂ O _{8 etc.} ). The progress of the polymerization can be confirmed by the decrease in the absorption intensity in the ultraviolet absorption band based on the diene group. The particle size does not change before and after the polymerization.

In addition to the above, the polymerizable phosphatidic acid of the present invention can be used to form a thin film by polymerizing the polymeric material on the surface of the polymeric material to impart appropriate medical suitability.

〔Example〕

Next, the present invention will be described with reference to an example that specifically describes the method for synthesizing the polymerizable phosphatidic acid of the present invention and a reference example that specifically describes the method for producing a polymer liposome from the polymerizable phosphatidic acid. Further description will be made.

Example 1 Synthesis of 1,3-bis (octadeca-trans, 2-trans, 4-dienoyl) glycerol (n = 12 compound of general formula (IV)); 9 ml (78 mmol) of quinoline and 1.7 g (18.5 m) of glycerol.
mol) was dissolved in 20 ml of dry chloroform and cooled to 10 ° C. To this, octadecadienoyl chloride (3
A solution of 6.25 mmol) dissolved in 10 ml of dry chloroform was slowly added dropwise (1 hour). After stirring overnight at room temperature, dry ether 1.5 was added, and the formed precipitate was filtered off. The filtrate was concentrated under reduced pressure, the residue was purified by silica gel chromatography (eluting solvent: chloroform), and the target portion was collected (thin layer chromatography (silica gel, hexane / ether =
1/1): Rf value = 0.3). After distilling off the solvent, the residue was recrystallized from hexane to obtain 2.3 g of the desired product (yield 20%).

IR (KBr): 1710cm ^-1 (ν c = o), 1650,1610cm ^-1 (ν
c = c); ¹ H-NMR (CDCl ³ , TMS) δ (ppm): 4.25 (4H, -CH ₂ -O
-), 2.60 5.80 (2H, -CO-CH =), 7.30 (2H, -COC = CH-), 6.16 (4H, -C = C
H-CH = C-); ¹³ C-NMR (CDCl ₃ , TMS) δ (ppm): 167.3 (ester carbonyl carbon); EIms: 616 (M ⁺ ); melting point 69-7
0 ° C; Elemental analysis value (% by weight): C 76.07 (75.92), H 11.
33 (11.11) (However, the value in parentheses is the calculated value of C ₃₉ H ₆₈ O _5. )

1,3-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-2-phosphoric acid (general formula (II
Synthesis of I) n = 12 compound); 0.24 ml (2.35 m) of phosphorus oxychloride in 10 ml of dry methylene chloride
(mol) was melted and cooled with ice. Separately, a solution prepared by dissolving 1.0 g (1.6 mmol) of the above glycerol derivative in 10 ml of methylene chloride and a solution prepared by dissolving 0.36 ml (1.65 mmol) of triethylamine in 10 ml of methylene chloride were separately and simultaneously prepared. It was slowly dropped into. After stirring at room temperature for 12 hours, 100 ml of dry benzene was added, and the formed precipitate was filtered off. The filtrate was concentrated under reduced pressure, 70 ml of tetrahydrofuran was added, and 0.5 M sodium hydrogencarbonate 5 was added.
0 ml and 25 ml of 0.25M (pH 10.8) ethylenediaminetetraacetic acid were added, and the mixture was stirred for 12 hours. Further, 50 ml of saturated saline was added, and the organic layer was collected and dried under reduced pressure. Chloroform / methanol / water = 50 ml / 50 ml / 50 ml was added to the residue and the mixture was stirred and the organic layer was collected. After distilling off the solvent under reduced pressure,
30 ml of chloroform was added to the residue and the insoluble portion was removed. The chloroform solution was dried and azeotropically treated with dry benzene. Dissolve the residue in 8 ml of chloroform and dry acetone 10
After adding 0 ml and centrifuging, the precipitate was collected, washed with acetone, and then vacuum dried over phosphorus pentoxide to obtain 0.5 g of the desired product (yield 41
%) Was obtained.

^{IR (KBr): 1720cm -1 (} ν c = o), 1650,1610cm -1 (ν
c = c), 3400,1260,1160,1100 cm ^-1 (phosphate); ¹ H
-NMR (CDCl ³ , TMS) δ (ppm): 4.25 (4H, -CH ₂ -O-), 2.60 5.80,7.30,6.16 (2H, 2H, 4H, -CH = CH-CH = CH-); ¹³ C-NMR
(CDCl ₃ , TMS) δ (ppm): 167.3 (ester carbonyl carbon); FABms: 763 (M + Na) ⁺ , 741 (M + H) ⁺ (C ₃₉ H ₆₇ O ₈ P ₁ Na ₂ molecular weight 740) UV absorption spectrum (CHCl ₃ ) λmax 261nm,
εmax 52500 (/ mol ・ cm); Thin layer chromatography (CHCl ₃
/ MeOH / water = 65/25/4) Rf value = 0.3; elemental analysis value (wt%): C61.7 (61.72), H9.01 (9.17) (however, the value in parentheses is C ₃₉ H ₆₇ Calculated value for O ₈ P ₁ Na ₂ · H ₂ O).

Examples 2 to 4 In the same manner as in Example 1, compounds of general formula (III) with n = 10,8,6 were synthesized. The analysis results are summarized in Table 1.

Example 5 Synthesis of 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycerol (n = 12 compound of general formula (VII)); 3-tritylglycerol 10.0 g (11.6 mmol) dried Dissolve in 200 ml of methylene chloride and cool to 0 ° C. To this was added 9.7 ml of BF ₃ methanol, and the mixture was stirred for 1.2 hours. To this, ether
After adding 300 ml, the mixture was ice-cooled, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. After adding 100 ml of hexane to the residue and cooling at 5 ° C. for 3 hours, the precipitate was collected, washed with cold hexane and dried in vacuum on phosphorus pentoxide.
5.7 g (yield 80%) of the desired product was obtained.

Thin layer chromatography (silica gel, ether / hexane = 1/1) Rf value = 0.35; EIms: 617 (M + H) ⁺ ; ¹³ C-NMR (CD
Cl ₃ , TMS): 167.3, 166.8 (ester carbonyl carbon), 14
6.3,145.7,128.2,118.3 (-C = CC = C-), 72.4,62.1,61.5
(Glycerol skeleton), 14.1 (Methyl), 22.7, 28.7, 2
9.2 to 29.7,31.9 (methylene CH _{2 n} ); Elemental analysis value: C
75.87 (75.92), H 11.21 (11.11) (However, the values in parentheses are
Calculated for C ₃₉ H ₆₈ O ₅ ).

1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-3-phosphoric acid (general formula (VI)
In the same manner as in Example 1, using 1.2 g (1.94 mmol) of 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycerol. Bis (octadeca-trans, 2-trans, 4-
Dienoyl) glycero-3-phosphoric acid was synthesized. Yield 48%.

Thin layer chromatography (silica gel, CuCl ₃ / MeOH / water = 6
5/25/4) Rf value = 0.22; FABms: 741 (M + H) ⁺ (C ₃₉ H ₆₇ O ₈ PiNa ₂
Has a molecular weight of 740); IR (KBr): 1705 cm ^-1 (ν c = o), 3350,126
0,1135,1100 cm ^-1 (phosphate); ¹³ C-NMR (CDCl ₃ / CD ₃
OD, TMS) δ (ppm): 167.8,167.6 (ester carbonyl carbon), 146.5,146.0,128.4,118.5 (-C = CC = C-), 70.6,6
3.0, 63.7 (glycerol skeleton), 14.2 (methyl), 23.
0,29.0 to 29.9,32.2,33.5 (methylene CH _{2 n} ); Elemental analysis value (% by weight): C 61.9 (61.72), H 9.20 (9.17) (However, the value in parentheses is C ₃₉ H ₆₇ O ₈ Calculated value of P ₁ Na ₂ · H ₂ O).

Example 6 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-3-phosphoric acid (formula (VI)
(N = 12 compound) (enzymatic method); 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-3-phosphatidylcholine (general formula (II)
(N = 12 compound) was dissolved in 15 ml of ether, and then 1
MCaCl ₂ 3.75 ml, 0.2 M sodium acetate buffer (pH 5.8) and phospholipase D solution extracted from cabbage (F.
M. Davidson, Biochemical Journal, Volume 69,
Pp. 458 (1958)) was added, and the mixture was vigorously stirred on a 37 ° C bath for 24 hours. 0.5M ethylenediaminetetraacetic acid (pH 8.
0) After adding 5 ml of the solution, chloroform / methanol (2
1) 50 ml, and then extracted twice with 50 ml of chloroform. The organic layers were combined and ice-cooled and then centrifuged to collect the organic layers. After the solvent was distilled off under reduced pressure, 30 ml of chloroform / methanol / 3% aqueous ammonia (6/5/1) was added to the residue for neutralization. After distilling off the solvent under reduced pressure, chloroform / methanol (1/1) is added to the residue to remove the insoluble portion. After evaporating the solvent, the residue was vacuum dried over phosphorus pentoxide to give the desired product (yield 45%).
Got The analytical value was in agreement with the compound of Example 5.

Reference Example 1 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-3-phosphatidylcholine (general formula (II)
N = 12 compound) 0.20 g (0.26 mmol), 1,3-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-2-
Phosphoric acid (n = 12 compound of general formula (III)) 0.095 g (0.13 mmol), cholesterol 0.20 g (0.52 mmol)
Was dissolved in benzene / methanol = 15 ml / 2 ml and lyophilized. 30 ml of 25 mM Tris buffer (pH 7.4) was added under an argon gas atmosphere, and ultrasonication was performed (probe type, 80 w, 30 minutes). The liposome solution (4 ml) was irradiated for 10 hours under argon gas in a quartz cell with a 100w low-pressure mercury lamp in a water bath at 8 ° C. Polymerization was confirmed by the disappearance of absorption in the characteristic absorption band (255 nm) based on the diene group. Polymer liposomes having an average particle size of 30 nm were obtained.

Reference Example 2 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-3-phosphatidylcholine (general formula (II)
N = 12 compound) 0.20 g (0.26 mmol), 1,2-bis (octadeca-trans, 2-trans, 4-dienoyl) glycero-2-
Phosphoric acid (n = 12 compound of general formula (VI))
Polymer liposomes were obtained in the same manner as in Reference Example 1 using 0.19 g (0.26 mmol) and cholesterol 0.10 g (0.26 mmol). (Average particle size 35 nm).

〔The invention's effect〕

Since the polymerizable phosphatidic acid of the present invention has a negative charge,
When this is used as a polymerized liposome or as a surface-treated thin film of a medical polymer, it is possible to significantly reduce the interaction with biological components.

Claims (2)

[Claims] 1. A general formula (However, R and R ^{1 in the} formula are (N is 6,8,10,12) an acyl group, Y is hydrogen or an alkali metal ion (Na ⁺ , K ⁺ ), and X is () Represents a polymerizable (α- or β-) phosphatidic acid 2. R and R ¹ are (Octadeca-trans, 2-trans, 4-dienoic acid), The polymerizable (α- or β-) phosphatidic acid according to claim 1.