JPH0141604B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a prophylactic and therapeutic agent for thrombosis.

Thrombosis is a pathological phenomenon in which platelets aggregate and blood coagulates in a living body's heart or blood vessels, forming a clot, or thrombus. The formation of this thrombus causes narrowing and occlusion of the vascular lumen. , causing ischemic lesions and infarctions in major organs such as the heart, brain, and lungs, resulting in dysfunction of these organs and causing clinically significant diseases. The causes of thrombus formation have not been clearly elucidated until now, but basically changes in the properties of the blood vessel wall, changes in blood flow, and changes in blood components are considered to be the factors, and recently, coagulation factors, Platelet adhesion and aggregation factors such as soluble factors and prostaglandins, and reticuloendothelial system functions are also attracting attention as being involved in the above-mentioned thrombus or intravascular blood coagulation. However, thrombosis occurs when the various factors mentioned above are abnormally closely and intricately intertwined, and since blood coagulation (coagulation) is observed in blood vessels in both cases, thrombosis cannot be used as a preventive or therapeutic agent. , drugs that act on the reaction system of the blood coagulation process, inhibit the coagulation mechanism, remove factors involved in this, and reduce the coagulability of blood, that is, blood anticoagulants (anticoagulants) are used. It's here. Representative examples thereof include sodium citrate, heparin, coumarin derivatives, and indandione derivatives. Considering their mechanism of action, these anticoagulants do not have the effect of dissolving blood clots that have already been produced, and therefore are intended to improve the hypercoagulable state and prevent the occurrence or recurrence of thrombosis. It is only effective for prevention. In recent years, enzyme preparations such as urokinase and streptokinase have been developed as thrombolytic agents that actively dissolve blood clots and improve blood flow. It has come to be widely used. Furthermore, it has been pointed out that the formation of the above-mentioned thrombi is closely related to platelet hyperfunction, and platelet function inhibitors, so-called antiplatelet agents, such as platelet membrane stabilizers and adenyl cyclase activators are used for the prevention and treatment of thrombosis. , phosphodiesterase inactivators, aspirin, non-steroidal anti-inflammatory drugs, etc.
LASS (Labile aggregation stimulating
Substance production inhibitors and aggregation inhibitors have been suggested, but their effectiveness has not yet been confirmed. Furthermore, it has recently been reported that eicosapentaenoic acid (EPA) is effective in preventing and treating thrombosis (Japanese Patent Application Laid-Open No. 154533/1983), but its preventive and therapeutic effects are still not satisfactory.

The present inventors have identified the mechanisms of action of the various known prophylactic and therapeutic agents for thrombosis, and in the process of intensive research into the blood coagulation mechanism, fibrinolytic mechanism, etc. Certain highly unsaturated fatty acids and their derivatives, which differ from various drugs, exhibit inhibitory effects on blood coagulation, or platelet aggregation, induced by arachidonic acid, adenosine diphosphate (ADP), and collagen, respectively. , also shows the effect of dissolving platelet aggregates, and its effect is more than twice that of EPA.
Therefore, it has been found to be effective in the prevention and treatment of thrombosis. The present invention was completed based on this new knowledge.

That is, the present invention provides (all-Z)-4, 7, 10, 13,
The present invention relates to a thrombosis preventive and therapeutic agent containing as an active ingredient at least one selected from 16,19-docosahexaenoic acid and pharmacologically acceptable salts, esters, and amides of the acid.

The prophylactic and therapeutic agent for thrombosis of the present invention is based on the use of the above-mentioned specific highly unsaturated fatty acids and derivatives thereof as an active ingredient, and can be used to treat deep vein thrombosis, extremity artery thrombosis, embolism, pulmonary embolism, retinal vein thrombosis, etc. disease,
It can be effective in preventing and treating various thromboses such as coronary artery thrombosis and cerebral thrombosis, as well as myocardial infarction, acute heart failure, stroke, etc. caused by these. In particular, the above-mentioned active ingredient compound of the present invention has good absorbability from the intestinal tract, can be taken internally, is stable in the blood, and therefore has a long duration of effect, and can be used in large amounts or for a long period of time. It can be taken and does not cause obesity due to excess calories.

(all-Z)-4,7,10,13,16,19-docosahexaenoic acid, which is used as one of the active ingredients in the present invention, is typically contained in a considerable amount in aquatic animal oil. It can be isolated from animals by conventional methods such as molecular distillation, countercurrent distribution, chromatography, etc., and some are commercially available as standards. However, for practical purposes, the above-mentioned highly unsaturated acids derived from aquatic animals do not need to be particularly isolated and purified pure products, and may be crude products containing a small amount of other highly unsaturated acids. Further, the above compound may be organically synthesized using appropriate starting materials. In the present invention, pharmacologically acceptable salts, esters, and amides of the above compounds can also be used as active ingredients in the same manner as the above compounds. The pharmacologically acceptable salts and esters typically include alkali metal salts such as sodium salts, potassium salts, calcium salts, and aluminum salts;
Examples include salts of alkaline earth metals and other metals, ammonium salts, amine salts such as morpholine, piperazine, trimethylamine and diethylamine, and lower alcohol esters such as methyl ester and ethyl ester.

Although the active ingredient compound of the prophylactic and therapeutic agent for thrombosis of the present invention can be administered alone, it is usually administered in the form of a pharmaceutical composition together with a pharmaceutical carrier. As carriers, fillers, extenders, binders, wetting agents, disintegrants,
Examples include diluents and excipients such as surfactants and lubricants. As the dosage unit form of the pharmaceutical composition, various forms can be selected depending on the purpose, and typical examples include tablets, pills, powder solutions, suspensions, emulsions, granules, capsules, suppositories, and injections. agent, (liquid agent,
suspending agents, etc.). When forming into a tablet, carriers include excipients such as lactose, sucrose, sodium chloride, glucose solution, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid, water, ethanol, propanol,
Single syrup, glucose, glycol, glycerin, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binders such as polyvinylpyrrolidone, starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, Disintegrants such as Twin, sodium lauryl sulfate, stearic acid monoglyceride, lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc., absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate, glycerin, starch Moisturizers, starch, lactose, kaolin, etc.
Adsorbents such as bentonite and colloidal silicic acid, and lubricants such as purified talc, stearate, boric acid powder, macrogol, and solid polyethylene glycol can be used. When forming into a pill form, carriers include excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, and laminaria. , agar, etc. can be used. Furthermore, the tablets can be made into conventionally coated tablets, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets, if necessary. When forming into a suppository, for example, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. can be used as carriers. When prepared as an injection, solutions and suspensions are preferably sterilized and isotonic with blood; when forming these solutions, emulsions, and suspensions, water may be used as a diluent, such as water. , ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, etc. can be used. In this case, the preparation may contain a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution. Further, when forming the composition into a paste, cream or gel form, white vaseline, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicone, bentonite and the like can be used as diluents. Furthermore, the thrombosis prevention and treatment agent of the present invention contains antioxidants such as butylated hydroxytoluene, propyl gallate, quinone, α-tocopherol, etc., and conventional solubilizing agents, buffers, soothing agents, and preservatives. Ingredients, colorants, fragrances, flavors, sweeteners, etc. and other pharmaceutical agents may be included, and certain antioxidants may promote anti-thrombotic effects.

The amount of the active ingredient compound to be contained in the pharmaceutical composition is not particularly limited and can be appropriately selected within a wide range, but it is usually 1% by weight or more based on the total composition, and in the case of tablets, for example, on a free acid equivalent weight basis. Approximately per tablet
Contains 0.2-1g of active ingredient compound.

Furthermore, the thrombosis prevention and treatment agent of the present invention is not particularly limited in its use, and can be administered in a manner suitable for various forms. For example, tablets, pills, solutions, suspensions,
Emulsions, granules, and capsules are administered orally; injections are administered alone or with glucose,
It is administered intravenously by mixing with normal replacement fluids such as amino acids, and if necessary, it is administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally, and in the case of suppositories, it is administered rectally, and in the case of women. may be administered intravaginally. The dosage of the preparation is appropriately selected depending on the administration method, patient's symptoms, etc., and is generally about 10 to 50 mg/Kg/day of the active ingredient, preferably 20 to 40 mg/Kg/day in terms of free acid weight. This is usually administered in 3 to 4 divided doses per day.

Furthermore, the above-mentioned active ingredient compound of the present invention can also be ingested by a patient in the required amount in the form of glyceride as margarine, butter, cooking oil, fat, etc. Therefore, the present invention provides such specific oils and fats. The present invention also provides a food form of a thrombosis preventive and therapeutic agent containing the following.

Thus, according to the present invention, an unprecedented agent for preventing and treating thrombosis is provided.

The present invention will be explained below with reference to experimental examples.

Experimental Example 1 <Reagent> (all-Z)-4,7,10,13,16,19-docosahexaenoic acid, which is the active ingredient compound (test compound) of the present invention, was obtained from Wako Pure Chemical Industries, Ltd. 500μg/ml, 250μg/ml and
Use as a 125 μg/ml ethanol solution. For comparison, EPA is used as a 500 μg/ml ethanol solution.

Collagen used as a platelet aggregation inducer includes:
Hormone The collagen reagent Horm, manufactured by Hormon Chemie (West Germany), was used in the form of a physiological saline solution, and ADP and arachidonic acid, manufactured by Sigma, were used in a physiological saline solution and ethanol, respectively. Use as a solution.

<Preparation of platelet-suspended plasma> 3.8% sodium citrate (0.1 times the blood volume) was added as an anticoagulant through a catheter inserted into the carotid artery of a Japanese white rabbit under non-anesthetized condition, blood was collected, and the blood was centrifuged. Separator (Kubota KN-70 type)
After centrifugation for 10 minutes at 1100 rpm in the same vessel, separate the supernatant, centrifuge the sediment in the same vessel for 15 minutes at 3000 rpm, separate the supernatant, and transfer this to a PPP (platelet
poor plasma). The number of platelets in the plasma obtained by centrifugation at 1100 rpm was measured using a Coulter counter ZB-1 type, and
PRP (platelet rich plasma) is prepared by diluting with PPP to a platelet count of 5×10 ⁸ cells/ml.

<Platelet aggregation test> Nature, 194 , 927-929,
(1962)], a platelet aggregation test is performed using an aggregometer manufactured by Niko Kizai Co., Ltd. ("aggregometer model PAT-6M type") according to the nephelometric method of Born et al. i.e. PRP200μ
into the measurement cuvette of the above aggregometer, and add 1μ of ethanol solution of test compound or EPA.
Add 1μ of ethanol and control solution, preincubate at 37℃ for 1 minute or 5 minutes, and adjust to the desired final concentration (for ADP).
7.5 μM, 20 μg/ml for collagen, and 50 μg/ml for arachidonic acid) were added to each of 20μ of ADP solution prepared with physiological saline or ethanol, 20μ of collagen solution, and 1μ of arachidonic acid solution. Causes aggregation. By continuously recording the turbidity change (absorbance change) of PRP using the aggregometer, the platelet aggregation inhibitory effect of the test compound or EPA at each concentration is investigated.

<Results> The results are shown in FIGS. 1 to 6. Figures 1 to 3 show ADP (1st
Figures 4 to 6 show the inhibitory effect of test compounds or EPA on platelet aggregation induced by collagen (Figure 2) and arachidonic acid (Figure 3), respectively, and Figures 4 to 6 show that the preincubation time was 1 min. FIG. 3 is a diagram showing a similar platelet aggregation inhibiting effect when In each figure, the horizontal axis shows time and the vertical axis shows absorbance change. In addition, curve 1 in each figure is the test compound concentration of 50μ
In the case of g/ml, curve 2 is 250μg/ml, curve 3 is 250μg/ml.
is 125 μg/ml, curve 4 is the control solution (no test compound and EPA added, control), and curve 5 is EPA
These are the respective data at a concentration of 500 μg/ml.

From Figures 1 to 6 above, especially Figures 1 to 3, the preferred concentration of the active ingredient compound of the present invention against any platelet aggregation induced by ADP, collagen, and arachidonic acid is Although it is different, it is clear that it has a sufficient blocking effect. From the above results and other various experimental results, it is clear that all the active ingredient compounds of the present invention are effective as preventive and therapeutic agents for thrombosis.

Experimental Example 2 Using the sodium salt of (all-Z)-4,7,10,13,16,19-docosahexaenoic acid in the form of a 500 μg/ml physiological saline solution, ADP, collagen, etc. The inhibitory effect of the above sodium salt on platelet aggregation induced by arachidonic acid was also investigated.

The results when the preculture time was 5 minutes were almost the same as in Figures 1 to 3, respectively, and the results when the preculture time was 1 minute were the same as in Figures 4 to 6, respectively. .

Experimental example 3 (all-Z)-4,7,10,13,16,19-docosahexaenoic acid ethyl ester at 500μg/ml
A test similar to Experimental Example 1 was conducted using the ethanol solution in the form of an ethanol solution.

The results of the inhibitory effect on ADP-induced platelet aggregation are shown in FIG. 7, the results of the inhibitory effect on collagen-induced platelet aggregation are shown in FIG. 8, and the results of the inhibitory effect on arachidonic acid-induced platelet aggregation are shown in FIG. 9, respectively. In each figure, curve 1 represents the test compound,
Curve 4 shows the control solution, respectively.

These figures show that the compound of the present invention in the form of ethyl ester also has excellent platelet aggregation inhibiting activity.

Experimental example 4 (all-Z)-4,7,10,13,16,19-docosahexaenoic acid amide was used as the test compound.
It was used in the form of a 500 μg/ml ethanol solution in the same manner as in Experimental Example 1.

The results are shown in Figures 10-12. 10-12 correspond to the above-mentioned FIGS. 6-9, respectively, and curves 1 and 4 in each figure also mean the same thing. In each figure, the preculture time is 5 minutes.

From FIGS. 10 to 12 above, it can be seen that the compounds of the present invention in the form of acid amide also exhibit excellent platelet aggregation almost as good as those in the form of free acids (FIGS. 1 to 3) and methyl esters (FIGS. 6 to 9). It has been found to have an inhibitory effect and is therefore effective as a prophylactic and therapeutic agent for thrombosis.

Example 5 This experiment was carried out according to the method of Hornstra [Brit. J. Haemotol., 19 , 321 (1970)]. i.e. Wistar rat (male, 250-350
g) is anesthetized with 50 mg/Kg of pentobarbital intraperitoneally, the abdomen is opened, a circulation path is created in the abdominal aorta using a polyethylene tube, a part of which is exposed outside the body, and the abdomen is closed. Approximately 24 hours after the above surgery, the test animal is tied to a fixed table, and a filter (20 μm, inner diameter 13 mm) is attached to its extracorporeal circulation path. The extracorporeal circuit was filled with physiological saline containing heparin (200-400 units) and kept in a constant temperature bath kept at 36±1°C. A saline solution of 1 μg/ml of ADP is administered from the front of the filter over a period of about 10 seconds to cause platelet aggregation. The pressure before and after the filter is measured, and the difference is calculated as an indicator of the degree of aggregation.

After administering ADP three times every 30 minutes, the above pressure difference was calculated to determine the aggregation degree of the control, and then the same as used in Experimental Example 1 as the test compound (all-Z)
A suspension of 4,7,10,13,16,19-docosahexaenoic acid in gum arabic is administered orally at an amount of 100 mg/Kg of active ingredient. One hour after this oral administration, the pressure difference before and after the filter is checked again to determine the degree of aggregation, and this is compared with the degree of aggregation of the control to determine the inhibition rate of the test compound against ADP-induced platelet aggregation. As a result, it was found that the compound of the present invention inhibited platelet aggregation by an average of 20% compared to the control.

Manufacturing example 1 Tablets are prepared, each having the following composition.

(all-Z)-4,7,10,13,16,19-docosahexaenoic acid 140 mg Starch 31.4 mg Lactose 125 mg Polyvinylpyrrolidone 1.8 mg Magnesium stearate 1.8 mg Total 300 mg

[Brief explanation of drawings]

Figures 1 to 12 show the inhibitory effects of the compounds of the present invention on platelet aggregation induced by ADP, collagen, and arachidonic acid, respectively.
These are graphs showing changes over time in the turbidity (absorbance) of blood samples used in experimental examples. In each figure, 1 indicates the test compound concentration of 500 μg/ml, 2 indicates the same concentration of 250 μg/ml, and 3 indicates the same concentration of 125 μg/ml. , 4 is control solution (control)
and 5 is a comparative compound with an EPA concentration of 500 μg/ml.
The following cases are shown below.

Claims (1)

[Claims] 1 Contains as an active ingredient at least one selected from (all-Z)-4,7,10,13,16,19-docosahexaenoic acid and pharmacologically acceptable salts, esters, and amides of the acid. A thrombosis prevention and treatment agent characterized by: