JPH0739373B2 - Method for producing high-purity eicosapentaenoic acid triglyceride - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for producing high-purity eicosapentaenoic acid triglyceride, and more particularly to a method for producing high-purity eicosapentaenoic acid triglyceride which can be used as a platelet aggregation inhibitor in fat transfusion. Is.

[0002]

2. Description of the Related Art Eicosapentaenoic acid (hereinafter sometimes abbreviated as EPA), which is a polyunsaturated fatty acid having 20 carbon atoms and 5 double bonds, causes platelet aggregation. It has been known that it has an inhibitory action and therefore is effective in treating and preventing thrombotic diseases caused by platelet aggregation, such as myocardial infarction and cerebral thrombosis.

This EPA is contained in natural fish oils such as sardine oil, saury oil, mackerel oil and the like in the form of free acid or triglyceride. However, since natural fish oil contains a large amount of various unsaturated fatty acids having carbon numbers or double bonds close to each other, it is extremely difficult to concentrate fish oil so as to increase the EPA content. Kaisho 5
As described in JP-A 9-67241, the limit is to concentrate the EPA content to about 30%.

However, with such a purity, it is necessary to administer a large amount of such concentrated fish oil in order to effectively exert the platelet aggregation-inhibiting action, and therefore a large amount of fatty acids other than EPA exist in large amounts. There are various problems in using fish oil itself as a medicine, such as the fact that there is a risk of over-nutrition and side effects in some cases, due to the fact that calories are given. Therefore, high-purity eicosapentaenoic acid triglyceride has been desired, but an effective production method thereof has not been developed.

Thus, the object of the present invention is to provide a method for producing high-purity eicosapentaenoic acid triglyceride which contains EPA and can exert an effective inhibitory effect on platelet aggregation with a small amount of administration, and can be favorably used as a platelet aggregation inhibitor. It is what

[0006]

Therefore, the present invention has a purity of 8%.
Reacting 0% or more of eicosapentaenoic acid with a condensing agent,
Then, glycerol is added and the reaction is performed in the presence of imidazole sodium to provide a high-purity eicosapentaenoic acid triglyceride.

In general, triglycerides of fatty acids are represented by the following formula when fatty acid residues are represented by R ¹ , R ² and R ³ .

[0008] In the case of eicosapentaenoic acid triglyceride, the fatty acid residues R ¹ , R ² and R ^{3 in} the above formula are all eicosapentaenoyl groups (EPA groups). However, in the case of the present invention, the glyceride which is an eicosapentaenoyl group together with the three fatty acid residues is mainly used, but any two or one of these three groups is an eicosapentaenoyl group. It also includes glycerides in which the other one or two are other fatty acid residues.

In the present invention, the high-purity eicosapentaenoic acid triglyceride means that the ratio of the amount of EPA groups to the total amount of fatty acid residues of the above eicosapentaenoic acid triglyceride is 80% or more, preferably 90% or more.

The method for producing high-purity eicosapentaenoic acid triglyceride (EPA-TG) according to the present invention will be described in detail. This is a high-purity EPA, for example, JP-A-57-187397 or JP-A-58-187. 8
80 produced by the method described in Japanese Patent No. 037
% Or more, preferably 90% or more of high-purity EPA and glycerol are reacted at room temperature in the presence of a condensing agent. For example, first, EPA and N,
An N'-carbonylimidazole condensing agent is reacted in a solvent under an inert gas such as nitrogen gas to give an acylimidazole, which is then treated with glycerol in a solvent using imidazole sodium obtained from imidazole and sodium hydride as a catalyst. When reacted, the starting material E used
Highly pure eicosapentaenoic acid triglyceride corresponding to the purity of PA can be obtained. Examples of the solvent used here include anhydrous tetrahydrofuran, anhydrous dimethyl sulfoxide, anhydrous acetonitrile, anhydrous benzene and the like.

The reaction for producing EPA-TG from EPA in this manner is shown in the formula below.

[0012]

[Chemical 1] A high-purity eicosapentaenoic acid triglyceride (EPA-TG) thus obtained, an emulsifier (egg yolk phospholipid, soybean phospholipid) and glycerol are dispersed in distilled water and emulsified under pressure to prepare a fat transfusion. can do. The amount is preferably 1 to 2 parts by weight of an emulsifier and 2 to 3 parts by weight of glycerol with respect to 10 parts by weight of EPA-TG, and preferably 100 parts by weight by adding the remaining amount of water.

According to the present invention, high purity eicosapentaenoic acid triglyceride (EPA-TG) is thus obtained.
Can be easily produced in high yield. The fat transfusion made from this is usually administered by intravenous injection. Thus, by intravenous administration, it can be administered even in an emergency case where oral administration cannot be performed, and platelet aggregation inhibition can be expressed earlier than when oral administration is performed. Moreover, since it contains high-purity eicosapentaenoic acid triglyceride, it is more effective when administered in a small amount than when using a low-purity one, and it does not cause overnutrition and has fewer side effects due to impurities mixed in in small amounts, thus suppressing platelet aggregation. Then, it can be effectively used for the treatment and prevention of thrombosis and the like. Moreover, it can be favorably used as a medicine without causing liver dysfunction or hyperlipidemia.

The EPA used as the raw material is fish oil such as sardine oil and mackerel oil, and fish oil and eventually marine resources can be effectively used.

[0015]

EXAMPLES The present invention will be further described below with reference to production examples of high-purity eicosapentaenoic acid triglyceride used for fat infusion of the present invention and test examples of fat infusion in order.
The eicosapentaenoic acids used as raw materials in Production Examples 1 and 2 are all 90% pure obtained by the method described in JP-A-57-187397. Production Example [1] 14 g (86.4) of N, N′-carbonyldiimidazole
mmole) was suspended in 40 ml of anhydrous tetrahydrofuran, and 21.8 g (72 mmol) of eicosapentaenoic acid was suspended in the suspension.
An anhydrous tetrahydrofuran solution (about 40 ml) of e) was added, and the mixture was stirred under a nitrogen stream at room temperature for 2 hours. To the above reaction solution, 20 ml of anhydrous dimethyl sulfoxide solution of 1.84 g (20 mmole) of glycerol was added, and further 20 ml of tetrahydrofuran solution of sodium imidazole {6 g of imidazole and 1.45 g of sodium hydride (about 50% in oil) were added in 20 ml of anhydrous tetrahydrofuran. What was prepared by reacting for 1 hour} and 1 ml of anhydrous pyridine were added, and the mixture was reacted under a nitrogen stream for 18 hours. Chloroform 2 was added to the obtained reaction solution.
00 ml was added, this was neutralized with 60 ml of 1N HCl, 100 ml of methanol was added, and the mixture was separated with a separatory funnel. The lower layer was separated and concentrated. The concentrate is dissolved in a hexane-ether (96: 4) mixture, passed through a column packed with 200 g of silica gel treated with the same solvent, and eluted with 6 l of the same solvent. The eluate is searched by thin layer chromatography to obtain 10.5 g of the target highly pure 90% eicosapentaenoic acid triglyceride (yield 55.3%).

This product is a thin layer chromatography [Silicage
A single spot was shown on a plate F254 E. Merck, developing solvent hexane-ether (85:15), detection reagent: iodine]. Mass spectrum of this product, nuclear magnetic resonance (N
The measurement results of the MR) spectrum and the infrared absorption (IR) spectrum are shown in FIGS. In mass spectrometry (Fig. 1), the molecular ion peak (M ⁺ ) was m / e 944 (C ₆₃ H ₉₂ O ₆ ).
Was recognized by.

¹ H-NMR spectrum δ value (CDC
l ₃ , ppm) (FIG. 2) δppm 0.95 (f, 9H) 1.2-2.5 (m, 24H) 2.82 (m, 24H) 4.1-4.3 (m, 5H) 5.1-5.6 (m, 30H) IR spectrum, cm ^-1 (NaCl cell, Neat)
(FIG. 3) 2960 (νas CH ₃ ) 1740 (νc = 0) 1660 (νc = c) 710 (δCH out-of-plane) Production Example [2] 12.85 g (7) of N, N′-carbonyldiimidazole
9.2mmole) and eicosapentaenoic acid 20.5g (6
6mmole) and dissolved in 30ml anhydrous acetonitrile,
Stir for 1 hour at room temperature under a stream of nitrogen. To this reaction solution, 20 ml of anhydrous dimethyl sulfoxide solution of 1.84 g (20 mmole) of glycerol was added, and further 20 ml of imidazole sodium dimethyl sulfoxide solution {5 g of imidazole and 1.44 g of sodium hydride (50% in oil,
30mmole) in anhydrous dimethylsulfoxide (20ml)
Prepared by reacting for 1 hour with 5 ml of pyridine and 5 ml of pyridine are added, and the mixture is stirred under a nitrogen stream for 3 hours at room temperature. Hexane-1N-HCl-methanol mixture (300
ml: 100 ml: 100 ml) is added, and the layers are separated with a separating funnel. The aqueous layer is further extracted three times with 200 ml of hexane and combined with the previous hexane layer. The hexane layer was then saturated aqueous sodium hydrogen carbonate solution (100 ml) and saturated aqueous sodium chloride solution (200 ml).
Wash with. The hexane layer was concentrated, the concentrate was dissolved in a hexane-ether mixed solution (96: 4), and the mixture was passed through a column packed with 400 g of silica gel treated with the same solvent system, which was eluted with 8 l of the same solvent as a thin layer. Chromatography was performed to obtain 16.65 g of the desired 90% highly pure eicosapentaenoic acid triglyceride (yield 88
%). Test Example [I] 90% pure EPA-T obtained in the above production example
A fat transfusion containing G was prepared and intravenously administered to a rabbit to measure its platelet aggregation-inhibitory effect, which was similarly obtained by the method described in JP-A-59-67241. Comparisons were made with the results performed on a fat transfusion made from fish oil containing EPA-TG and a control. 1. Sample oil i) 90% EPA-TG ii) 30% EPA-TG iii) Soybean oil (trade name Infrafat) Control product 2. Infusion composition (100 ml) Sample oil 10 g Glycerol 2.5 g Egg yolk phospholipid 1.2 g Water Remaining infusion composition consisting of each sample oil, 5 to 6 rabbits weighing about 3 kg were used to collect blood before administration 3 days later
An infusion of 0 ml was intravenously injected, another 30 ml was intravenously injected on the 4th day, and blood was collected again on the 7th day.

Blood was collected in a syringe containing a small amount of sodium citrate to prepare platelet rich plasma (PRP) with a constant platelet count. The PRP of each rabbit was dispensed into a cuppet and kept in a furan vessel, and then an aggregation inducer was added, and then the light transmittance was measured using a nephelometer, thereby measuring the platelet aggregation rate.

When adenosine diphosphate (ADP) 5 μM was used as an aggregation inducer, collagen 1
The platelet aggregation rate was measured for each case using 0 μg / ml. Then, the average value of the platelet aggregation rate in the blood collected before administration and in the plasma collected on the 7th day was calculated and compared. [II] Next, in order to investigate the effect of each lipid transfusion sample on plasma lipids, the blood collected before administration and the dose were EP
90% EPA-TG is 30 ml each on the 1st and 4th day after blood collection so that the amount is almost equal in A equivalent.
After each 100 ml of EPA-TG was intravenously injected, the amount of neutral lipid and cholesterol in each plasma of blood collected on the 7th day was measured and compared. [III] In order to examine the effect on the liver, glutamic acid, pyruvic acid, transaminase enzyme (GP) in blood collected before administration and blood after administration are similarly measured.
The amount of T) was measured and compared. Since the amount of GPT increases when there is hepatocellular injury, the change in the amount before and after administration is measured.

The results are shown below.

[0021]

[Table 1]

[0022]

[Table 2]

[0023]

[Table 3]

[0024]

[Table 4]

[0025]

As is clear from the above results, according to the present invention, high-purity eicosapentaenoic acid triglyceride can be easily produced in a high yield, and the high-purity (90%) obtained by the present invention can be obtained. In the case of the EPA-TG infusion, administration of 30 ml × 2 times showed a significant decrease in platelet aggregation and a slight increase in blood lipids and GPT.

On the other hand, with 30% EPA-TG, there was no significant difference in the platelet aggregation ability before and after administration of 30 ml of the infusion solution twice, and therefore, the platelet aggregation inhibitory action was not shown. Thus, as 100 mg x 2 doses of 30% EPA-TG, which is almost equivalent to 90 ml EPA-TG 30 ml x 2 doses, increases in blood lipids and GPT are shown, and hyperlipidemia and liver function are shown. Obviously, there is a risk of injury.

Thus, when high-purity EPA-TG is used, administration of a smaller amount than that of low-purity EPA-TG exerts a good platelet aggregation inhibitory effect without increasing blood lipids and GPT. be able to. Moreover, for example, as an infusion solution having the above composition, an adult usually 100 to 300 once a day.
Since 0 ml, preferably 500 to 2000 ml, can be administered by intravenous injection, it can be administered even when it cannot be administered orally.
Moreover, the effect can be exhibited earlier than oral administration.
Thus, this infusion can be effectively used for the treatment and prevention of thrombotic diseases, and further for the prevention of cancer metastasis.

[Brief description of drawings]

FIG. 1 shows a mass spectrometric spectrum, a nuclear magnetic resonance spectrum, and an infrared absorption spectrum of high-purity eicosapentaenoic acid triglyceride obtained by Production Example 1 of the present invention.

FIG. 2 shows a mass spectrometry spectrum, a nuclear magnetic resonance spectrum, and an infrared absorption spectrum of high-purity eicosapentaenoic acid triglyceride obtained by Production Example 1 of the present invention.

FIG. 3 shows a mass spectrometry spectrum, a nuclear magnetic resonance spectrum, and an infrared absorption spectrum of high-purity eicosapentaenoic acid triglyceride obtained by Production Example 1 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location C07B 61/00 300 (72) Inventor Yasuji Souda 4-5 Motoyama Kitamachi, Higashinada-ku, Hyogo Prefecture −11 (72) Inventor Akira Miyamoto 1-1-16 Takasu-cho, Nishinomiya-shi, Hyogo Prefecture 16-316 (72) Inventor Fujimi Nanano 12 Shinmachi, Hashimoto, Takatsuki-shi, Osaka

Claims (1)

[Claims] 1. A method for producing high-purity eicosapentaenoic acid triglyceride, which comprises reacting eicosapentaenoic acid having a purity of 80% or more with a condensing agent, and then adding glycerol and reacting in the presence of imidazole sodium.