JPH0717504B2 - Composition for preventing and treating thrombosis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for preventing and treating thrombosis, which comprises vitamin A or its ester and vitamin C as active ingredients.

Cerebrovascular diseases and heart diseases such as cerebral hemorrhage, cerebral thrombosis, cerebral infarction, heart failure and myocardial infarction are closely related to blood clotting and formation of thrombus due to a decrease in fibrinolytic system. Conventionally, in the prevention and treatment of these thrombosis, heparin, a coumarin derivative is used as a drug that suppresses the coagulation system, and urokinase, an enzyme preparation such as streptokinase has been developed as a drug that advances the fibrinolytic system. , Antigenicity, pyrogenicity, short duration, etc.

The present inventor focused on the fact that plasmin that dissolves fibrin that constitutes a thrombus is produced by the action of plasminogen activator produced from vascular endothelial cells on plasminogen, and searched for a substance that promotes the production of plasminogen activator. As a result, vitamin A or its ester and vitamin C
The present invention was made by discovering that a large amount of plasminogen activator is released from vascular endothelial cells by the combined action of both when combined with and.

Vitamin A (retinol) is used as a medicine for night blindness and the like, and vitamin C (L-ascorbic acid) is used as a medicine for scurvy and the like, but its preventive and therapeutic effects on thrombosis are not known.

Examples of vitamin A esters include acetate, laurate, palmitate, stearate, etc.
Esters with _{12- C18} saturated fatty acids are preferred.

The agent for preventing and treating thrombosis of the present invention is administered as an oral or parenteral preparation by mixing vitamin A or its ester and vitamin C with itself or a pharmaceutically acceptable carrier. Oral formulations include powders, tablets, granules, capsules, solutions, emulsions and syrups. Examples of parenteral preparations include injections, drops, suppositories and the like. Examples of excipients that can be used include water, ethanol, lactose, starch, dextrin, calcium phosphate, calcium carbonate, aluminum silicate, magnesium oxide, magnesium stearate, dry aluminum hydroxide and the like.

The dose varies depending on the patient's age, medical condition, etc., but is 10,000 to 100,000 IU / day as vitamin A for adults and vitamin C50 to 10
00 mg / day is used.

The test examples of the present invention are shown and described below.

Test Example 1 Fresh bovine carotid artery was obtained from a slaughterhouse, and the carotid artery was incised to scrape the endothelial cell layer to obtain endothelial cells. These vascular endothelial cells were treated with 10% fetal bovine serum and streptomycin (50
mg / ml) and penicillin (50 units / ml) in a Petri dish containing Eagle's minimum essential medium at 37 ° C and 5% CO ₂
Cultured in a constant temperature bath. The cultured endothelial cells were cultivated for one generation in a Petri dish in the same medium as described above, containing a predetermined amount of vitamin A and vitamin C dissolved in ethanol. After culturing, the medium was removed, the endothelial cells were washed with a serum-free minimum essential medium of Eagle, and then incubated in the same serum-free medium at 37 ° C for 8 hours to remove the plasminogen activator released from the endothelial cells. An endothelial cell sample solution containing the same was prepared and stored at -20 ° C.

A fibrin suspension for measuring the plasminogen activating activity of this sample solution was manufactured as follows. 0.1 ml 3
0 μM fibrin monomer solution was dissolved in 1M sodium bromide-0.05M acetate buffer at pH 5.3 to form fibrin clots, 2 ml 100 g / l gum arabic, 3 ml 0.5M imidazole buffer (pH 7). .5) and 25 ml of 5 mM phosphoric acid-0.15 M
A mixture of Nacl buffer (pH 7.5) was added. This liquid is 20kHz
Sonicate for 30 minutes to produce a fine particle fibrin suspension and store at 4 ° C.

The plasminogen activation activity was measured by the following method. 700 μl fibrin suspension prepared above and 100 μl
200 μl of the above-mentioned endothelial cell sample solution was added to a mixed solution of 0.7 units of plasminogen, and the mixture was kept at 37 ° C., and the decrease in turbidity of the fibrin suspension was measured by a nephelometer. The standard curve was prepared from the value at which the turbidity was reduced by 20% in the same manner except that 0.005-0.08 units of urokinase was used instead of the sample solution. The plasminogen activating activity in the sample solution was shown in the unit of urokinase conversion per 10 ⁸ endothelial cells by comparing the value at which the turbidity was reduced by 20% with the standard curve.

The plasminogen activating activity of the specimen liquid obtained by incubating endothelial cells cultured by adding vitamin A and vitamin C alone or at the same time for 8 hours is as shown in Table 1.

When only Vitamin A is cultivated with varying the amount added, increasing the amount of Vitamin A added increases the plasminogen activation activity, and at high concentrations of 10 μM or higher, it shows a constant value at a level about 10 times higher than without addition. It was

On the other hand, when only Vitamin C was cultivated while changing the addition amount thereof, the release amount of the plasminogen activator showed a constant value at a level about twice that of no addition, by adding about 100 μM or more of Vitamin C.

However, when vitamin A and vitamin C are added at the same time, first, the amount of vitamin C added is fixed at 50 μM, and when the amount of vitamin A is changed and cultured, increasing the amount of added vitamin A results in plasminogen activating activity. Increased with it, when using vitamin A 10 μM and vitamin C 50 μM together, about 15 times as much as adding vitamin C 50 μM alone,
The activity value was about 25 times higher than that without addition.

In addition, the amount of vitamin A added was fixed at 10 μM and vitamin C
When the amount of Vitamin C was changed and the amount of Vitamin C added was increased, the plasminogen activating activity also increased accordingly.
The release amount of the plasminogen activator increased dramatically, about double that of the case without addition.

Also, vitamin A palmitate 10 μM and vitamin C 150
The same test was also performed for the combination with μM, and as shown in Table 2, the amount of plasminogen activator released was greatly increased.

From Test Example 1 described above, it can be seen that the combined use of vitamin A or its ester and vitamin C clearly shows a summing effect and releases a large amount of plasminogen activator.

Test Example 2 Ethanol 1 containing Vitamin A 1000 IU and Vitamin C 30 mg
1 ml of the 1% solution was injected into the ear vein of a female rabbit weighing about 3 kg.
2 ml of blood was collected from the ear vein over time, and the amount of fibrinogen in serum and the amount of FDP, which is a fibrin degradation product, were
Quantitation was performed using a spectroscopic method (Clim. Chem., 24 , 351, 1978) and a commercial FDP kit, respectively. As a result, the amount of fibrinogen in blood decreased with the passage of time, while the amount of FDP increased conversely, and the opposite correlation was established between the two. That is, after 6 hours, the amount of fibrinogen in blood was reduced by about 30% from the normal state, and the FDP amount was as high as 15 μg / ml, which was 10 times that in the normal state.

As is clear from the above test examples, the agent for preventing and treating thrombosis of the present invention stimulates vascular endothelial cells to produce a plasminogen activator, which is released extracellularly to activate the fibrinolytic system. Therefore, the effect of preventing thrombus formation or accelerating the melting of fibrin clots for treatment is remarkable, and is superior to conventional enzyme preparations without fear of antigenicity.

Claims (1)

[Claims] 1. Vitamin A or its ester and vitamin C
A composition for preventing and treating thrombosis, which comprises as an active ingredient.