JPH0840981A - Eicosapentaenoyl glyceride - Google Patents

Abstract

PURPOSE:To obtain a new compound useful for treating and preventing various diseases derived from hyperlipemic serum, having excellent lowering action on lipid in serum and inhibitory action on blood platelet aggregation. CONSTITUTION:This compound is expressed the formula (at least two of R<1> to R<3> are eicosapentaenoyl and the rest is H) such as trieicosapentaenoyl glyceride. The compound of the formula is obtained by halogenating eicosapentaenoic acid of the formula, R-COOH (X is a halogen) to give an eicosapentaenoic acid halogenide of the formula, R-COX (X is a halogen) and reacting the compound with glycerol. This new compound of the formula has low toxicity and excellent absorption by oral administration and is effective for treating preventing asthmatic attack, diabetes, arthritis, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel eicosapentaenoyl glyceride, more specifically, the following general formula (1)

[0002]

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(Wherein at least two of R ¹ , R ² and R ³ represent eicosapentaenoyl groups, and the rest represent hydrogen atoms).

[0004]

2. Description of the Related Art Conventionally, the problems to be solved by the invention
Eicosapentaenoic acid represented by the following formula (2) (hereinafter,
It is known that “EPA”) has an effect of lowering plasma cholesterol in the human body and can be used for prevention or treatment of thrombosis.

[0005]

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[0006] Further, EPA is contained as a glyceride in marine oils and fats such as mackerel, sardines and cod, and this glyceride is a triglyceride composed of one EPA and two other fatty acids. Therefore, when such a natural triglyceride is administered in an effective amount of EPA, a large amount of calories are provided due to other fatty acids, resulting in overnutrition.

Therefore, in order to overcome such drawbacks, a triglyceride composed of two EPAs and one other fatty acid has been proposed in recent years (Japanese Patent Laid-Open No. 59-1909).
No. 48). However, this still has one fatty acid, and the above problems have not been completely solved.

[0008]

In such an actual situation,
As a result of intensive studies to solve the above problems, the present inventor has found that diglyceride and triglyceride of EPA are well absorbed in oral administration and show excellent lipid-lowering action and platelet aggregation-inhibiting action. Completed the invention.

That is, the present invention provides eicosapentaenoyl glyceride (hereinafter referred to as "EPAG") represented by the above general formula (1).

The EPA-G of the present invention includes the following.

Trieicosapentaenoyl glyceride [EPA-TG]

[0012]

[Chemical 4]

1,2-di (eicosapentaenoyl) glyceride [1,2-EPA-DG]

[0014]

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1,3-di (eicosapentaenoyl) glyceride [1,3-EPA-DG]

[0016]

[Chemical 6]

The EPAG of the present invention is produced, for example, by halogenating EPA (2) into an EPA-halogenide (3) according to the following reaction formula, and reacting this with glycerin.

[0018]

[Chemical 7]

(R is an eicosapentaenoyl group, and X is
Indicates a halogen atom, R¹, R², R ³Means the above
Have)

The raw material EPA is, for example, JP-A-58-80.
A highly pure product can be obtained by decomposing EPA-ethyl ester obtained by the method described in No. 37 with ethanol and caustic potash. The halogenation of EPA can be carried out by a method known per se. For example, if oxalyl chloride is used as a halogenating agent and the reaction is carried out at a temperature of 65 to 90 ° C. for 4 hours, EPA-
Chloride is obtained. The reaction between EPA-halogenide (3) and glycerin is carried out by heating under reflux in a solvent such as chloroform in the presence of a base such as quinoline or pyridine for several hours. EPA-halogenide is used in an amount 3 times or more the molar amount of glycerin. The reaction product thus obtained is a mixture of EPA-TG, 1,2-EPA-DG and 1,3-EPA-DG, which is subjected to silica gel column chromatography as shown in Examples below. Therefore, it can be separately obtained as EPA-TG and EPA-DG (mixture of 1,2-form and 1,3-form). And these ratios are about 1: 1.

The physical properties of EPA-TG and EPA-DG are as shown in Table 1.

[0022]

[Table 1]

[0023]

Next, the biological activity of EPA-TG and EPA-DG will be shown.

Experiment 1 Acute toxicity (mouse) (1) Experimental method EPA-TG or EPA-D was used, with 10 SIC: ICR mice of 4 weeks old having a body weight of 25 to 30 g per group.
Oral (po) or intraperitoneal (ip) administration of G, 7 after administration
It was observed for a day and examined for acute poisoning symptoms and lethal effects during that period. (2) Experimental results Oral administration: EPA-TG or EPA-DG in both female and male
No one died within 7 days after 25 g / kg po, and diarrhea was observed 1 day after administration for acute toxic symptoms, but it returned to normal 3-4 days after administration, and other changes I couldn't see it. Intraperitoneal administration: EPA-TG 10 g / kg i for both female and male
None of them died within 7 days after p, and none of them showed marked acute poisoning symptoms. (3) Conclusion The lethal dose in mice of EPA-TG and EPA-DG is 25 g / kg or more in the case of oral administration (po) in both female and male, and 10 g / kg or more in the case of intraperitoneal administration (ip). is there.

Experiment 2 Hypolipidemic action in serum (rat) (1) Experimental method A group of 7 male SD rats weighing about 200 g was used.
EPA-TG 0.5g / kg or EPA-DG 0.75g
/ Kg, safflower oil 5.0 g / kg, and purified water 5.0 g / kg as a control for 30 days each by oral administration (p
After o), blood was collected and the lipid concentration in serum was measured. The lipid concentration in serum was measured by an automatic analyzer (Hitachi 705) using the following measuring reagents. Total cholesterol: Cholesterol 705 "Nissui"
(Nissui Pharmaceutical) Phospholipid: Phospholipid-705 "Nissui" (Nissui Pharmaceutical) Triglyceride: Triglyceride-705 "Nissui" (Nissui Pharmaceutical) Free fatty acid: NEFA-C set "Nissui" (Nissui Pharmaceutical) (2) ) Experimental results As shown in Table 2, EPA-TG 0.5 g / kg and EP
A-DG 0.75 g / kg po significantly reduced the total cholesterol and phospholipid concentrations in serum. On the other hand, with 5.0 g / kg of safflower oil, no marked changes were observed in the total cholesterol and phospholipid concentrations. Serum triglyceride concentration was the same in both EPA-TG and EPA-DG groups and safflower oil administration group, and serum free fatty acid concentration was EP.
The A-TG and EPA-DG groups and the safflower oil group showed a decreasing tendency as compared with the control group.

[0026]

[Table 2]

Experiment 3 Serum lipid lowering action in hyperlipidemic animals (rat) (1) Experimental method High-cholesterol shown below 2 weeks before the start of administration of the test drug was prepared with 10 male Wistar rats per group. While allowing food to be ingested, the test drug was orally administered (po) every day for 8 weeks, and then blood was collected, and the total cholesterol concentration and free cholesterol concentration in serum were measured by the same method as in Experiment 2.

[0028]

Table 3 Formulation of high cholesterol diet Milk casein 18.0 (%) Sucrose 61.0 Cellulose 4.0 Hardened vegetable oil 10.0 Minerals 4.0 Vitamins 2.0 Cholesterol 0.5 Sodium cholate 0.0. 5

(2) Experimental results As shown in Table 4, EPA-TG 6 mg / kg or EPA-D
G10 mg / kg po markedly suppressed the increase of serum total cholesterol and free cholesterol levels due to intake of a high-cholesterol diet. The serum free cholesterol concentration in the normal diet group was also decreased by po of EPA-TG 60 mg / kg or EPA-DG 90 mg / kg.

[0030]

[Table 4]

Experiment 4 Platelet Aggregation Inhibitory Action (Rabbit) (1) Experimental Method A diet in which healthy male rabbits weighing about 2.0 kg were grouped into 4 groups and 0.5% of cholesterol was added (Oriental RC- It was bred according to 4). EPA-TG is 200 mg /
kg and EPA-DG 300 mg / kg were orally administered (po) every day for 4 weeks. After 4 weeks, blood was collected from the ear vein, platelet-rich plasma (PRP) was prepared, and the platelet aggregation ability by 1.25 μg / ml collagen was measured using “Aggregometer” (AUTORAM-21 type) manufactured by Rika Denki. did. (2) Experimental Results As shown in Table 5 and FIG. 3, the platelet aggregation ability was significantly suppressed in the EPA-TG or EPA-DG administration group as compared with the untreated group.

[0032]

[Table 5]

Experiment 5 Preventive Effect on Asthma Attacks (1) Experimental Method EPA-TG 0.9 g / day was applied to the remaining 3 cases in 5 out of 8 cases of male asthma between the ages of 30 and 55 with no seasonality. EPA-DG 1.5 g / day was orally administered daily in the form of soft capsules, and the effect on the occurrence of asthma attack was examined. (2) Experimental results In the case of EPA-TG, 1 out of 5 treated patients was E.
I had been taking prednin 5mg daily for 4 months before the start of PA-TG administration, but the number of asthma attacks started to decrease 1 month after the start of EPA-TG administration, and I was able to withdraw from predonin after 3 months. It was Another example is EPA-TG
Nearly three months after the start of administration, the number of attacks decreased significantly. In the remaining 3 cases, no change was observed in the occurrence of asthma attack even after continuous administration of EPA-TG for 3 months, and the administration was discontinued. In the case of EPA-DG, 2 out of 3 patients have EPA-
The number of episodes of asthma attacks decreased 1 month after the start of TG administration, and after 3 months almost no attacks occurred. In the remaining 1 patient, the tendency of seizure onset did not decrease even after the administration for 3 months, so the administration was discontinued. This allows EPA-T
It was found that G and EPA-DG have a remarkable preventive effect on the occurrence of asthma attack.

Experiment 6 Preventive Effect on Experimental Nephritis (Rat) (1) Experimental Method A group of 10 6-week-old male Donryu rats was used, and the method of Glassock et al. [Journal of Experimental. Medicine (J. Exp. M
ed. 127 , 555 (1968)], Heymann nephritis was prepared, and the preventive effect of EPA-TG and EPA-DG on the development of this nephritis was examined.
The water-insoluble fraction of the renal tube of Donryu rat 20 mg / individual was injected with 0.5 ml of Freund's complete adjuvant into the rat footpad subcutaneously on both hind limbs for sensitization. From the day after sensitization, EPA-TG or EPA- 6 DG 100 mg / kg
Oral administration was carried out every day every week, and changes in urinary protein excretion were examined.
Purified water 0.1 ml / kg was administered to the control group in the same manner as the test drug. Urine protein amount Nagamatsu et al's method [day pharmacological magazine 81, 29
5 (1983)]. (2) Experimental results As shown in Table 6, EPA-TG and EPA-DG100
The administration of mg / kg significantly suppressed the excretion of urinary protein 6 and 8 weeks after the sensitization. This allows EPA-TG and E
PA-DG was found to have a protective effect on the progression of autoimmune complex nephritis.

[0035]

[Table 6]

Experiment 7 Effect on Experimental Diabetes (Rat) (1) Experimental Method Five male Donryu rats weighing about 200 g each were dissolved in a citrate buffer solution of pH 4.5 for the diabetes onset group. Streptozotocin 30 mg / kg was injected intravenously. The test drug is 24 hours before streptozotocin IV, 1
Oral administration was performed before the time and after 5 hours. Venous blood was collected 24 hours after intravenous injection of streptozotocin, and an automatic analyzer (Hitachi 70) was used using a reagent V-GLU-C set "Nissui" (Nissui Pharmaceutical Co., Ltd.) for measuring glucose by an enzymatic method.
6 type). (2) Experimental results As shown in Table 7, EPA-TG or EPA-DG10
Oral administration of 0 mg / kg significantly suppressed streptozotocin diabetes. This allows EPA-TG and EPA-
It was found that DG has a preventive effect on the onset of streptozotocin diabetes and an inhibitory effect on the progress thereof.

[0037]

[Table 7]

Experiment 8 Effect on Adjuvant Arthritis (Rat) (1) Experimental Method Seven-week-old female Donryu rats were bred for 1 week and used for the experiment. Freund's complete adjuvant 0.
Adjuvant arthritis was induced by injecting 06 ml into the tail skin of the rat. The test drug was orally administered once a day for 21 days from the day before the adjuvant injection. The evaluation of the arthritis condition was performed by the arthritis score for the extremities and both pinna 2 and 3 weeks after the adjuvant injection. (2) Experimental results As shown in Table 8, EPA-TG or EPA-DG100
Oral administration of mg / kg significantly suppressed the arthritis score 2 and 3 weeks after the adjuvant injection. This makes E
It was found that PA-TG and EPA-DG have a preventive effect on the progressive development of adjuvant arthritis.

[0039]

[Table 8]

[0040]

As is apparent from the above experiments, the eicosapentaenoylglyceride (1) of the present invention has extremely low toxicity, has an excellent serum lipid-lowering action and a platelet aggregation-inhibiting action, and has a high fat content. It is effective in treating and preventing various diseases derived from serum.

[0041]

EXAMPLES Next, examples will be described.

Example 1 40 g (0.121 mol) of EPA-ethyl ester and 10% KOH ethanol [8.15 g as KOH]
(0.145 mol)] and charged with N ₂ gas (15
(0 ml / min), and refluxed at 75-76.5 ° C for 1 hour. The reaction product was cooled to room temperature, adjusted to pH 2 with 10% HCl water, and an inorganic salt was precipitated, so 50 ml of water was added to dissolve it, and the mixture was extracted twice with 100 ml and 50 ml of n-hexane. The extract was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure at 40 ° C. to obtain 35.9 g of oily EPA (yield 98.1%).

Example 2 To 35.1 g (0.116 mol) of EPA, 29.5 g (0.232 mol) of oxalyl chloride was added dropwise at room temperature while introducing N ₂ gas. Then, the mixture was reacted at 65 to 75 ° C. for 4 hours, and after the reaction, excess oxalyl chloride was distilled off with an evaporator. Distill the residue under reduced pressure to 144 ° C / 1mm
Fractions of Hg to 187 ° C./2 mmHg were collected to obtain 18.78 g (yield 50.4%) of EPA-chloride.

Example 3 Glycerin 1.81 g (0.0197 mol), quinoline 10.8 g (0.084 mol) and chloroform 80.
1 g was charged, and 18.0 g of EPA-chloride was added.
(0.056 mol) was gradually added dropwise. 72 to 8
Refluxed at 0 ° C. for 3.5 hours. After cooling the reaction solution, it was poured into 540 ml of petroleum ether to prepare a 0.5N-sulfuric acid aqueous solution 300
ml was added with stirring, stirred for 10 minutes, and allowed to stand for 30 minutes.
The layers were separated, and 300 ml of a 5% aqueous potassium carbonate solution was added to the upper layer to separate the layers. 300 ml of water was added to the upper layer for liquid separation, 100 ml of saturated saline was further added for liquid separation, and the upper layer was collected. This was dried over anhydrous magnesium sulfate and petroleum ether was distilled off with an evaporator to obtain 15.15 g of an oily substance.
I got A glass column was filled with 500 g of silica gel using special grade benzene, and 10 g of the above oily substance dissolved in 100 ml of special grade benzene was added thereto. Next, the same benzene (8 L) was used for elution, and 300 ml fractions were collected. About this fraction, TLC (Kieselgel 60F
₂₅₄ , developing solvent: benzene, confirmation: I ₂ vapor), the eluate was confirmed, the same dissolved water was collected, and the solvent was distilled off under reduced pressure to obtain the following substance.

[0045]

[Table 9]

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of EPA-TG.

FIG. 2 is a diagram showing an infrared absorption spectrum of EPA-DG.

FIG. 3 is a graph showing the platelet aggregation inhibitory effect of EPA-TG and EPA-DG.

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Claims (1)

[Claims] 1. The following general formula (1): (In the formula, at least two of R ¹ , R ² and R ³ represent eicosapentaenoyl groups, and the rest represent hydrogen atoms), an eicosapentaenoyl glyceride.