JP2016155820A - Antithrombotic agents specifically inhibiting intrinsic blood clotting reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop antithrombotic agents specifically inhibiting formation or growth of pathologic thrombus without the side effect such as bleeding.SOLUTION: The present invention relates to antithrombotic agents containing sacran that is an Aphanothece Sacram-derived sulfation polysaccharide, and to food and drinks containing the sacran extracted from Aphanothece Sacrum, the food and drinks having antithrombotic effect that inhibits intrinsic blood clotting reactions and does not inhibit extrinsic clotting reactions, as well as to food additives containing sacran.SELECTED DRAWING: None

Description

  The present invention relates to an antithrombotic agent. Specifically, the present invention relates to an antithrombotic agent that specifically inhibits an intrinsic blood coagulation reaction, including a cyanobacterium sulfated polysaccharide.

  Thrombosis such as cerebral infarction, myocardial infarction, and pulmonary embolism (economy class syndrome) often leads to death, and about 1/3 of Japanese death causes such a blood circulation disorder. Drugs for the prevention and treatment of thrombosis have been developed so far, but they all have side effects of bleeding. For example, even if cerebral thrombus can be prevented, death due to side effects of cerebral hemorrhage is not rare. .

  It is known that sulfated polysaccharides such as heparin inhibit blood coagulation (see Patent Document 1), and are already widely used in clinical and laboratory settings as antithrombotic agents and blood coagulation inhibitors at the time of blood collection. ing. Sulfated polysaccharides such as heparin prevent the coagulation reaction by catalytically enhancing the thrombin inhibitory activity of antithrombin. Therefore, these sulfated polysaccharides inhibit blood clotting reactions activated in both endogenous and exogenous systems. In particular, heparin is a very strong antithrombotic agent, and suppresses pathological thrombus formation, but also inhibits physiological thrombus formation (hemostatic action), so the side effect of bleeding is inevitable.

Majerus, PW; Tollefsen, DM (2006), “Blood coagulation and anticoagulant, thrombolytic, and antiplatelet drugs”, in Laurence L. Brunton, Goodman & Gilman's the pharmacological basis of therapeutics (11th ed.), McGraw-Hill Companies

  The problem to be solved by the present invention was to develop an antithrombotic agent that does not inhibit hemostasis but inhibits pathological thrombus formation.

  The present inventors have made extensive studies to solve the above-mentioned problems, and by using a sulfated polysaccharide sacran derived from the cyanobacterium Aphanothece Sacrum, hemostasis is not inhibited, and only pathological thrombus formation is inhibited. The inventors have found what can be done and have completed the present invention.

That is, the present invention provides the following:
(1) an antithrombotic agent containing a cherry or a cherry-containing material, which inhibits an intrinsic blood coagulation reaction and does not inhibit an exogenous blood coagulation reaction,
(2) Food or drink having an antithrombotic effect that inhibits the intrinsic blood coagulation reaction and does not inhibit the extrinsic blood coagulation reaction, and (3) food addition containing cherry or cherry-containing material Agent.

  The antithrombotic agent of the present invention specifically suppresses an intrinsic blood coagulation reaction involved in pathological thrombus formation and does not inhibit an exogenous coagulation reaction involved in physiological thrombus formation (hemostatic action). That is, the antithrombotic agent of the present invention specifically inhibits the formation and increase of pathological thrombus, but has no side effect of bleeding, and thus can be said to be an ideal antithrombotic agent. Moreover, according to the manufacturing method of this invention, the food / beverage which has the above ideal antithrombotic effects is provided.

FIG. 1 is a graph showing the results of examining the effect of sulfated polysaccharides on the intrinsic blood coagulation reaction. FIG. 2 is a graph showing the results of examining the influence of sulfated polysaccharides on the extrinsic blood coagulation reaction. FIG. 3 is a graph showing the results of examining the influence of sulfated polysaccharides on antithrombin concentration-dependent thrombin inhibitory activity. FIG. 4 is a graph showing the results of examining the effect of high-concentration cherry on thrombin inhibitory activity by antithrombin.

  In one aspect, the present invention provides an antithrombotic agent that inhibits an intrinsic blood coagulation reaction and does not inhibit an extrinsic blood coagulation reaction, containing cherry or a cherry-containing material.

  Endogenous blood coagulation is initiated by the activation of factor XII by the binding of macromolecular kininogen and plasma kallikrein on negatively charged substances. The extrinsic blood coagulation reaction is initiated when a coagulation factor VIIa in the blood forms a complex with tissue factor of a membrane protein existing outside the blood vessel. Existing antithrombotic agents suppress both intrinsic and extrinsic blood clotting reactions, and it is impossible to suppress only the intrinsic blood clotting reaction, and it is easy to bleed or bleed Side effects such as difficulty in hemostasis have been a problem. Since the antithrombotic agent of the present invention specifically inhibits the intrinsic blood coagulation reaction and does not inhibit the exogenous blood coagulation reaction, there is no fear of the above side effects. In this respect, the antithrombotic agent of the present invention can be said to be ideal.

  In the present specification, the antithrombotic agent means an agent for suppressing or preventing thrombus formation in a subject, or an agent for treating or preventing thrombosis.

  Sakuran is an extracellular matrix produced by the cyanobacterium Aphanothece sacrum. Cherry is a sulfated polysaccharide and has a molecular weight of about 20 MDa. Constituent monosaccharides contained in cherry are various, such as glucose, galactose, mannose, xylose, rhamnose, fucose, galacturonic acid, glucuronic acid, galactosamine, muramic acid, and a sulfate group is added to about 11% of sugar residues. ing. Cherry is characterized by containing sulfated muramic acid.

  Heparin and heparan sulfate have a repeating structure of iduronic acid or glucuronic acid and glucosamine. In addition, the seaweed sulfated polysaccharide fucoidan such as mozuku, which the present inventors have clarified the antithrombotic action, mainly comprises fucose (see Patent No. 4428486). While such sulfated polysaccharides have a simple structure, as described above, cherry is composed of a very wide variety of monosaccharides. Considering the combination of monosaccharide type and sulfation site, Sakuran is expected to have a very diverse and unique sugar chain sequence. Thus, cherry may inhibit the intrinsic clotting reaction by a completely new molecular mechanism not found in previous sulfated polysaccharides.

  The cherry can be extracted from the swallowtail using a known method. For example, it can be obtained by extracting from suzenzinori using an aqueous sodium hydroxide solution.

  Sakura is known to have high water absorption and is applied to cosmetics. Sakuran has also been found to be used as a drug delivery carrier (Japanese Patent Laid-Open Nos. 2014-133809 and 2011-068606). However, it has never been known that cherry can be used as an antithrombotic agent.

  The cherry used in the antithrombotic agent of the present invention may be a crude product extracted from cyanobacteria, typically extracted from suizendinori, and is further purified using a known method such as chromatography. May be.

  The cherry-containing material used in the antithrombotic agent of the present invention is preferably a highly safe material that can be used in medicines and foods. The saccharin-containing material is exemplified by a group of Suizendinori, but may be a group of other cyanobacteria as long as it produces cherries having the above-described characteristics. Further, the cherry-containing material may be processed into various shapes, for example, processed into a plate shape, a sheet shape, a granule shape, a powder shape, a paste shape, a solution shape, or the like.

  The antithrombotic agent of the present invention can be preferably administered to mammals including humans in the form of a pharmaceutical composition. Examples of the administration route of the antithrombotic agent of the present invention include oral administration, intravenous injection, subcutaneous injection, and the like, but are not limited thereto. The antithrombotic agent of the present invention may be mixed with food and drink and given to the subject.

  The dosage form of the antithrombotic agent of the present invention may be any dosage form. For example, for oral administration, it can be formulated into various oral dosage forms such as concentrates, powders, granules, tablets, capsules, drinks and the like. For injection, for example, it can be in the form of an ampoule, a vial, an infusion bottle, or the like. Methods for producing these dosage forms are known, and processes such as mixing, dissolution, pulverization, tableting, and drying can be used. The preparation can also be carried out by appropriately using carriers and excipients according to the purpose. The antithrombotic agent of the present invention may be provided as a supplement.

  The dosage of cherry by the antithrombotic agent of the present invention can be appropriately determined by a doctor in consideration of the health condition, symptoms, age, weight, pre-existing condition, etc. of the subject. The dose for oral administration to an adult may be, for example, about 100 mg or more per day, or about 1 g or more per day. The frequency of administration of the antithrombotic agent of the present invention may be once to several times a day, or may be once to several times a few days. The antithrombotic agent of the present invention can be used prophylactically and therapeutically. The antithrombotic agent of the present invention may be used in combination with other antithrombotic agents. In the case of heparin, concomitant monitoring with other antithrombotic agents requires careful monitoring to increase bleeding tendency, but Sakuran is unlikely to promote bleeding and is considered desirable with other antithrombotic agents Conceivable. Other antithrombotic agents include vitamin K-dependent coagulation factor synthesis inhibitors such as warfarin, direct thrombin such as dabigatran, rivaroxaban, edoxaban, and apixaban, factor Xa inhibitors, aspirin, ticlopidine, clopidogrel, prasugrel, beraprost Examples thereof include, but are not limited to, antiplatelet drugs such as cilostazol, dipyridamole, dirazep, eicosapentaenesan, and thrombolytic drugs such as urokinase and t-PA.

  In a further aspect, the present invention provides a method for treating or preventing thrombosis in a subject, characterized by administering sakuran or a cherry-containing material to the subject, which inhibits an intrinsic blood coagulation reaction and exogenous blood coagulation. A method is provided that does not inhibit the reaction.

  In a further aspect, the present invention provides the use of cherry or a cherry-containing material for the manufacture of an antithrombotic agent that inhibits the intrinsic blood clotting reaction and does not inhibit the extrinsic blood clotting reaction.

  The present invention provides, in a further aspect, the use of cherry or a sacran-containing material for the treatment or prevention of thrombosis in a subject, which inhibits the intrinsic blood clotting reaction and does not inhibit the extrinsic blood clotting reaction To do.

  In a further aspect, the present invention provides a food or drink containing a cherry or a cherry-containing material. The food or drink containing the cherry or the cherry-containing material of the present invention is preferably a food or drink having an antithrombotic effect that inhibits the intrinsic blood coagulation reaction and does not inhibit the extrinsic blood coagulation reaction.

  The content of cherries in the food and drink of the present invention can be determined as appropriate, but preferably, antithrombosis that inhibits the intrinsic blood clotting reaction by eating food and drink on a daily basis and does not inhibit the extrinsic blood clotting reaction. An effective amount of cherry is added to the food and drink. Examples of added amounts of cherry include about 1% to less than 100%, about 5% to less than 100%, or about 10% to less than 100%, such as about 2%, about 5%, about 10%, about 20 %, About 50% (weight% as dry cherry).

  The food and drink of the present invention can be in any form. For example, it can be in the form of beverages such as soup and juice, pellets, granules, powders and the like. For example, it may be in the form of a supplement such as a drink, tablet or granule. Moreover, it can also be set as the form which adds and uses the food / beverage of this invention to the existing food / beverage.

  The food or drink of the present invention can be produced by adding sakura or a cherry-containing material in any production process of food or drink, adding it to a raw material of food or drink, or adding it to the produced food or drink. For example, you may use the cherry blossoms extracted from the suizenjinori for manufacture of food and drink. The cherry blossoms extracted from Suizenjinori may be refine | purified and does not need to be refine | purified. The extraction method of cherries from Suizenjinori is known and can be performed using various methods. For example, when using the alkali extraction method, the sardine is extracted as it is or after freezing-thawing treatment to remove pigments, etc., and then immersed in a 0.1 M aqueous sodium hydroxide solution and stirred at 100 ° C. for 4 hours to extract cherry. can do. Thereafter, neutralization, washing with an organic solvent such as acetone or ethanol, or purification by chromatography such as gel filtration may be appropriately performed. Subsequently, the extracted cherry is added in the manufacturing process of food and drink. The extracted cherry may be added to the raw material of food and drink, or may be added in the raw material processing step. Moreover, you may add the extracted cherries to the already completed food and drink.

  You may use the swamp genus colony as it is, or it may dry suitably, cut into pieces, or grind | pulverize, and may use it for manufacture of the food / beverage products of this invention.

  In a further aspect, the present invention provides a food additive comprising a cherry or a cherry-containing material. Preferably, the food additive of the present invention is used for producing a food or drink having an antithrombotic effect that inhibits the intrinsic blood coagulation reaction and does not inhibit the exogenous blood coagulation reaction. The form of the food additive of the present invention may be the same as the form of a normal food additive, and is not particularly limited. For example, it may be a liquid, paste, powder, granule, pellet, or the like. The food additive of the present invention can be produced by known means / methods such as extraction, mixing, pulverization, drying, concentration, and tableting.

  Hereinafter, the present invention will be described in more detail and specifically with reference to examples. However, the examples should not be construed as limiting the present invention.

(1) Purification and isolation of cherry from Suizendinori Suizenjinori was freeze-thawed and washed with pure water to remove reddish violet pigment. The washed Suizendinori was washed with a large amount of ethanol by shaking (120 rpm, 3 times overnight). Filtration was performed using gauze to obtain an ethanol-washed suizenjinori. Ethanol-washed suizendinori was added to 0.1 M NaOH aqueous solution and stirred vigorously at 100 ° C. for 4 hours to obtain a clear solution. The clear solution was neutralized with HCl to pH 8.0-9.0 and filtered. The filtrate containing cherry was slowly poured into 100% acetone to obtain a white fibrous precipitate. The white fibrous precipitate was redissolved in hot water and reprecipitated. Redissolution and reprecipitation were repeated three times. The precipitate is dissolved in pure water to make an aqueous solution (pH of about 6.0 to 7.0), purified by gel filtration chromatography (Sephadex LH-20, GE Healthcare), and a fraction of a clear solution containing cherry is collected. The lyophilized product was used for the following experiments.

(2-1) Effect of sulfated polysaccharides on intrinsic and extrinsic coagulation reactions Diluted activated partial thromboplastin time (dAPTT) method as an in vitro blood coagulation test using human plasma Diluted prothrombin time (dPT) method (Takeya H., et al .: Anti-β2-Glycoprotein I (β2GPI) monoclonal antibodies with lupus anticoagulant-like activity enhance the β2GPI binding to phospholipids. J Clin Invest 99: 2260-2268 (1997)). In plasma, negatively charged substances ellagic acid and phospholipid were used as activators in the dAPTT method, and TF reconstituted with phospholipids was used as the activator in the dPT method. In both methods, first, an activator and a sample (sakuran, heparin, heparan sulfate) were added and incubated at room temperature for 5 minutes, and then 50 mM CaCl ₂ was added and a thrombin generation reaction was performed for 5 minutes. Thereafter, the reaction was stopped with EDTA, and the chromogenic substrate Z-Val-Pro-Arg-pNA or HD-Phe-Pip-Arg-pNA (S-2238) was added and incubated for 5 minutes. The color reaction was stopped with 20% acetic acid, and the absorbance at 405 nm was measured using a spectrophotometer (DU (registered trademark) 640 SPECTRO PHOTOMETER).

(2-2) Measurement of thrombin inhibitory activity by antithrombin Purified antithrombin (0 to 1.6 μg / mL) and a sample (cherrant 10 μg / mL, heparan sulfate 10 μg / mL, heparin 0.2 μg / mL) were added. After incubating at room temperature for 5 minutes, 0.1 μg / mL thrombin was added, incubated for 5 minutes, EDTA and chromogenic substrate Z-Val-Pro-Arg-pNA or HD-Phe-Pip-Arg-pNA (S- 2238) was added and incubated for 5 minutes. After stopping the reaction with 20% acetic acid, the absorbance at 405 nm was measured using a spectrophotometer. Further, the antithrombin concentration was fixed at 0.5 μg / mL, and the cherry concentration was changed to ˜40 μg / mL.

(2-3) Effect of Sakuran on Coagulation Time of Human Plasma Using human plasma, the effect of cherry on the intrinsic and extrinsic coagulation reactions was examined. The clotting time of human plasma was measured using a clotting time measuring reagent for clinical examination, and the effects of cherry and heparin were examined. Endogenous activated clotting time was obtained by measuring activated partial thromboplastin time (APTT) using actin FSL (actin FSL reagents, Siemerns Healthcare Diagnostics). The activation time of the extrinsic system was obtained by measuring the prothrombin time (PT) using Thromborel S (Siemens Healthcare Diagnostics). Experiments were performed according to the instructions for use of actin FSL and thromborel S, respectively.

The results are shown in Table 1. Heparin prolongs the clotting time of both APTT and PT, whereas Sakuran did not affect PT but prolonged APTT alone. That is, it was confirmed that cherry also specifically inhibits the intrinsic clotting reaction in human plasma, but does not affect the extrinsic clotting reaction.

(3) Results and Discussion Effects of Sulfated Polysaccharide Sakura on Exogenous and Endogenous Coagulation Reactions Sulfated polysaccharides such as heparin and heparan sulfate are produced by coagulation serine proteases such as thrombin and factor Xa and serpins such as antithrombin. Indirect (catalytic) promotion of complex formation inhibits the blood clotting reaction. Since the same anticoagulant action was assumed for cherry, first, the influence of cherry on the intrinsic coagulation reaction using ellagic acid as a negatively charged substance was examined. As a result, Sakuran was slightly weaker than heparin, but showed an inhibitory effect on the intrinsic coagulation reaction similar to that of heparan sulfate (FIG. 1). Then, next, when the influence of Sakuran on the extrinsic coagulation reaction starting from TF was examined, it was shown that Sakuran did not suppress thrombin generation at all unlike heparin and heparan sulfate (FIG. 2). Moreover, the thrombin activity inhibitory action by antithrombin was remarkably enhanced by heparin and heparan sulfate, whereas sacran had no effect (FIG. 3). A similar experiment was performed using a high concentration of cherry (40 μg / mL), but the high concentration of cherry showed no antithrombin activity enhancing action (FIG. 4). Furthermore, it was confirmed that Sakuran specifically inhibits the intrinsic clotting reaction in human plasma, but does not affect the exocoagulant clotting reaction. From these results, it was clarified that Sakuran specifically inhibits the intrinsic clotting reaction, but does not affect the extrinsic clotting reaction. Therefore, it was considered possible to develop an antithrombotic agent without side effects of bleeding by using cherry.

  The present invention can be used in the field of pharmaceuticals, particularly in the field of antithrombotic agents.

Claims (3)

  An antithrombotic agent containing a cherry or a cherry-containing material that inhibits an intrinsic blood coagulation reaction and does not inhibit an exogenous blood coagulation reaction.   A food or drink having an antithrombotic effect that inhibits an intrinsic blood coagulation reaction and does not inhibit an exogenous blood coagulation reaction, containing cherry or a cherry-containing material.   Food additive containing cherry or cherry-containing material.

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