KR102008826B1 - Pharmaceutical composition for inhibiting platelet aggregation or for thrombolysis comprising nattokinase and anti-inflammatory agents - Google Patents

Abstract

The present invention relates to a thrombolytic adjuvant pharmaceutical composition comprising an anti-inflammatory agent and a platelet aggregation inhibiting or thrombolytic pharmaceutical composition comprising a nattokinase and an anti-inflammatory agent as an active ingredient. The thrombolytic auxiliary pharmaceutical composition according to the present invention can effectively reduce the bleeding side effects of the conventional thrombolytic use by including dexamethasone as an active ingredient, and can be usefully used as a thrombolytic auxiliary pharmaceutical material. In addition, the pharmaceutical composition comprising nattokinase and dexamethasone as an active ingredient shows a prominent thrombolytic synergistic effect compared to the nattokinase alone component, so that even a low dose of nattokinase can lead to excellent thrombolytic activity. It can increase the human safety through, and with this has the advantage that can effectively reduce the side effects of bleeding.

Description

Pharmaceutical composition for inhibiting platelet aggregation or thrombolysis including nattokinase and anti-inflammatory agents as an active ingredient

The present invention relates to a thrombolytic adjuvant pharmaceutical composition comprising an anti-inflammatory agent and a platelet aggregation inhibiting or thrombolytic pharmaceutical composition comprising a nattokinase and an anti-inflammatory agent as an active ingredient.

Abnormal activation of the platelet aggregation system causes blood vessel thrombus formation, and thrombus generated in closed vessels such as arteries, veins or microvascular vessels prevents the smooth flow of blood in the blood vessels. Platelet adhesion and aggregation are major factors in vascular thrombosis, and thrombi can grow to a size sufficient to block arterial vessels. Blood clots may also form in stagnant or low blood flow velocity regions within the vein. Venous blood clots can easily detach from their location and travel through the circulatory system, resulting in occlusion of other blood vessels, such as the pulmonary artery. Thus, arterial thrombosis causes serious disease due to local obstruction, and venous thrombosis mainly causes distant occlusion, or embolism.

Thus, medications with platelet aggregation inhibition or thrombolytic action include those associated with blood clots, embolisms such as myocardial infarction, angina, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, peripheral vascular or deep vein thrombosis, and any Arterial thrombosis of veins, venous thrombosis, ischemic cerebral infarction, arteriosclerosis, hypertension, pulmonary hypertension, cerebral infarction, stroke, chronic arterial obstruction, pulmonary infarction, pathologic symptoms after embolism, renal embolism, thromboembolism and subarachnoid hemorrhage, percutaneous coronary angioplasty ( Thrombus formation during PTCA) and stent insertion, restenosis after stent placement, catheter thrombotic occlusion or reoccluding, acute coronary syndrome, TIA (transient cerebral ischemic attack or acute cerebrovascular syndrome), heart failure, ischemic etiology It can be used for the prevention and treatment of chest pain, syndrome X, diabetes mellitus.

In particular, thrombosis is one of the major diseases that endanger human health. For example, mortality due to thrombosis and acute myocardial infarction has exceeded 40%, making thrombosis the disease with the highest mortality. Thrombolysis is an important way to treat these diseases. However, clinical trial management criteria, such as streptokinase (SK), urokinase (UK) and tissue-type plasminogen activator (abbreviated as 't-PA' for short) The thrombolytic drugs currently used to treat thrombosis are all toxic, have serious side effects such as bleeding, have a short half-life in vivo, and are expensive. Indeed, thrombolytic dissemination via intravenous injection of tissue plasminogen activator (t-PA) is an approved medical treatment only for acute ischemic stroke, and 6.8% of patients have been reported to have cerebral hemorrhage following t-PA injection.

On the other hand, nattokinase is a profibrinolytic serine protease rich in natto, an oriental traditional food, produced by Bacillus subtilis Natto during soybean fermentation. Natokinase is known to lower blood pressure and prevent thrombosis. In addition, nattokinase can conserve blood flow by inhibiting thrombosis, and this effect has been suggested to be achieved through thromboxane formation and platelet aggregation inhibitory activity. Natokinase has been approved by the Korea Food and Drug Administration (KFDA) as a dietary supplement for improving blood circulation.

However, in the case of nattokinase, which is relatively safe compared to other thrombolytics, a large dose should be used to derive a thrombosis treatment effect, and there is a problem in that high doses of hemorrhagic side effects are used.

Therefore, the present inventors have tried to find a method that can improve the hemorrhagic side effects of the conventionally used thrombolytic agents, and as a result, when using anti-inflammatory dexamethasone (dexamethasone) in combination with the conventional thrombolytic agents, thrombolytic activity The present invention has been completed by confirming that while reducing the side effects of bleeding while improving.

Korean Patent Publication No. 10-2008-0008393 Korean Patent Publication No. 10-2017-0029755

It is therefore an object of the present invention to provide a thrombolytic supplemental pharmaceutical composition that can reduce bleeding side effects resulting from the use of thrombolytics.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting platelet aggregation or thrombolysis, which has reduced bleeding side effects.

It is still another object of the present invention to provide a pharmaceutical preparation for inhibiting platelet aggregation or thrombolysis with reduced bleeding side effects.

In order to achieve the above object,

The present invention provides an antithrombotic auxiliary pharmaceutical composition comprising an anti-inflammatory agent as an active ingredient.

In one embodiment of the invention, the anti-inflammatory agent may be dexamethasone (dexamethasone).

In one embodiment of the present invention, the thrombolytic agent may be nattokinase, tissue-type plasminogen activator, streptokinase, or urokinase.

In one embodiment of the present invention, the composition may increase the thrombolytic activity of thrombolytics and reduce bleeding side effects due to the use of thrombolytics.

The present invention also provides a pharmaceutical composition for inhibiting platelet aggregation or thrombolysis, comprising a thrombolytic agent and an anti-inflammatory agent as an active ingredient.

In one embodiment of the present invention, the thrombolytic agent may be nattokinase, tissue-type plasminogen activator, streptokinase, or urokinase.

In one embodiment of the invention, the anti-inflammatory agent may be dexamethasone (dexamethasone).

In one embodiment of the present invention, the dexamethasone (dexamethasone) can increase the thrombolytic activity of thrombolytics and reduce the bleeding side effects of the use of thrombolytics.

The present invention also provides a pharmaceutical preparation for inhibiting platelet aggregation or thrombolytic activity prepared from the composition.

In one embodiment of the present invention, the formulation may be selected from the group consisting of hard and soft capsules, tablets, dragees, film coated tablets, uncoated tablets, enteric tablets, powders, suspensions, syrups and injections.

The thrombolytic auxiliary pharmaceutical composition according to the present invention can effectively reduce the bleeding side effects of the conventional thrombolytic use by including dexamethasone as an active ingredient, and can be usefully used as a thrombolytic auxiliary pharmaceutical material. In addition, the pharmaceutical composition comprising nattokinase and dexamethasone as an active ingredient shows a significant thrombolytic effect of thrombosis compared to the nattokinase alone component, so that even a low dose of nattokinase can lead to excellent thrombolytic activity. Through it can increase the safety of the human body, and also has the advantage to effectively reduce the bleeding side effects.

Figure 1a is a graph showing the measurement of the blood flow rate (%) according to the treatment (intravenous injection) of nattokinase for carotid arterial thrombosis induced by FeCl ₃ (○: solvent PBS group, ▽: 25 mg / kg nattokinase Administration group, □: 50 mg / kg nattokinase administration group, ◇: 75 mg / kg nattokinase administration group, △: 100 mg / kg nattokinase administration group).
Figure 1b is a graph showing the measurement of the blood flow rate (%) according to the treatment (intravenous injection) of the tissue-type plasminogen activator (t-PA) for carotid arterial thrombosis induced by FeCl ₃ (○: solvent PBS administration group , ▽: 5 mg / kg t-PA group, □: 7.5 mg / kg t-PA group, ◇: 10 mg / kg t-PA group).
FIG. 2A is a graph showing the measurement of blood flow rate (%) according to nattokinase (25 mg / kg) alone or a combination of nattokinase and dexamethasone (intravenous injection) for carotid arterial thrombosis induced by FeCl ₃ (○: Solvent PBS group, ●: dexamethasone (2 mg / kg) alone group, ▽: nattokinase (25 mg / kg) alone group, ▼: nattokinase (25 mg / kg) and dexamethasone (2 mg / kg) combination group) .
FIG. 2B is a graph showing blood flow rate (%) according to nattokinase (50 mg / kg) alone or in combination with nattokinase and dexamethasone (intravenous injection) for carotid arterial thrombosis induced by FeCl ₃ (○ : Solvent PBS administration group, ●: Dexamethasone (2 mg / kg) administration group, □: Natokinase (50 mg / kg) administration group, ■: Natokinase (50 mg / kg) and dexamethasone (2 mg / kg) administration group ).
FIG. 2C is a graphical representation of measuring blood flow rate (%) following t-PA (5 mg / kg) alone or t-PA and dexamethasone combination treatment (intravenous) for carotid artery thrombosis induced by FeCl ₃ (FIG. ○: solvent PBS administration group, ▽: t-PA (5 mg / kg) alone administration group, ▼: t-PA (5 mg / kg) and dexamethasone (2 mg / kg) combined administration group).
FIG. 2D is a graphical representation of measuring blood flow rate (%) following t-PA (7.5 mg / kg) alone or t-PA and dexamethasone combination treatment (intravenous) for carotid arterial thrombosis induced by FeCl ₃ (FIG. ○: solvent PBS group, ●: dexamethasone (2 mg / kg) alone group, □: t-PA (7.5 mg / kg) alone group, ■: t-PA (7.5 mg / kg) and dexamethasone (2 mg / kg) ) Combination administration group).
Figure 3 is a result of observing the degree of arterial blockage caused by blood clots in the tissue following drug administration (intravenous injection) in FeCl _3- induced arterial thrombosis animal model (phosphate buffered saline (PBS), dexamethasone ( Dexa; 2 mg / kg), nattokinase (Natto; 25 mg / kg) or tissue plasminogen activator (t-PA; 5 mg / kg).
Figure 4 is a result of observation of hemorrhagic lesions in the tissues according to nattokinase 100-300 mg / kg administration (intravenous injection) in the arterial thrombosis animal model by visual and histological staining (A: lung, B: thymus, arrow: bleeding) ).
5 is a result of observation of hemorrhagic lesions in tissues according to t-PA 10-30 mg / kg administration (intravenous injection) in the arterial thrombosis animal model by visual and immunohistochemical staining (A: lung, B: thymus, Arrow: bleeding).

The present invention relates to a pharmaceutical composition for thrombolytic supplements comprising an anti-inflammatory agent as an active ingredient.

The present invention is based on the finding that dexamethasone, an anti-inflammatory agent, enhances the activity of known thrombolytic agents and reduces bleeding side effects resulting from the use of thrombolytic agents.

The inventors of the present invention used a combination of dexamethasone together in the arterial thrombosis animal model when compared to intravenous injection of nattokinase or tissue-type plasminogen activator (t-PA) alone as a thrombolytic agent. When treated, the bleeding response was weakened, and the safety margin was increased through experiments (see FIGS. 4-5 and Table 1). In addition, the blood flow rate was remarkably improved in the experimental group in which dexamethasone was combined with the experimental group administered with nattokinase alone in the arterial thrombosis animal model (see FIGS. 2A and 2B).

The pharmaceutical composition for thrombolytic aid of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant, in addition to the active ingredient dexamethasone, and the adjuvant may include an excipient, a disintegrant, a sweetener, and a binder. , Coating agents, expanding agents, lubricants, lubricants or flavoring agents may be used.

In addition, the pharmaceutical composition may be preferably formulated into a pharmaceutical composition (pharmaceutical composition), including one or more pharmaceutically acceptable carriers in addition to the active ingredient dexamethasone (dexamethasone) for administration.

The present invention also relates to a platelet aggregation inhibiting or thrombolytic pharmaceutical composition (pharmaceutical composition) comprising a thrombolytic and anti-inflammatory agent as an active ingredient.

In one embodiment of the present invention, the thrombolytic agent may be nattokinase, tissue-type plasminogen activator, streptokinase or urokinase.

In another embodiment of the invention, the anti-inflammatory agent is aspirin, diclofenac, indomethacin, sulindac, ketoprofen, flurobiprofen, child Borfurofen (iborprofen), naproxen, pyroxicam, tenoxicam, tolmetin, ketoraclac, oxaprosin, meso Mefenamic acid, fenprofen, nambumetone (Relafen), acetaminophen (tylenol), nimesulide, NS-398, flusulid, L -745337, celecoxib, rofecoxib, SC-57666, DuP-697, parecoxib sodium, JTE-522, valdecoxib, SC-58125 , Etoricoxib, RS-57067, L-748780, L-761066, APHS, tododolac, meloxicam, S-2474, hydrocortisone, coltizone (cortisone), prednisone (p rednisone, prednisolone, methylprednisolone, methylprednisolone, meprednisone, triamcinolone, paramethasone, fluprednisolone, dexamethasone, dexamethasone Fludrocortisone, desoxycorticosterone, and the like can be exemplified, but preferably dexamethasone.

In the present invention, when using dexamethasone (dexamethasone) in combination with a thrombolytic agent as an anti-inflammatory agent, it was confirmed through experiments that can increase the thrombolytic activity of thrombolytic agents and reduce the bleeding side effects of the use of thrombolytic agents.

Meanwhile, the pharmaceutical composition provided by the present invention is a pharmaceutical composition for inhibiting platelet aggregation or thrombolysis, and may be a pharmaceutical composition (pharmaceutical composition) capable of preventing or treating thrombotic disease.

In the present invention, "thrombotic disease" means a disease caused or promoted by a thrombus, for example, myocardial infarction, angina pectoris, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, peripheral vascular or deep vein thrombosis, arterial thrombosis , Venous thrombosis, ischemic cerebral infarction, arteriosclerosis, hypertension, pulmonary hypertension, cerebral infarction, stroke, chronic arterial obstruction, pulmonary infarction, cerebral embolism, renal embolism, thromboembolism and subarachnoid hemorrhage, percutaneous coronary angioplasty (PTCA) and stent thrombus formation at stent insertion, restenosis after stent placement, catheter thrombotic occlusion or reclosure, acute coronary syndrome, TIA (transient cerebral ischemic attack or acute cerebrovascular syndrome), heart failure, chest pain due to ischemic etiology, X Syndrome, diabetes mellitus can be selected from the group, but the type is not particularly limited.

In the present invention, "treatment", unless stated otherwise, refers to a disease or condition to which the term applies, or to reverse, alleviate, inhibit, or prevent the progression of one or more symptoms of the disease or condition. As used herein, the term "treatment" refers to the act of treating when "treating" is defined as above. Thus, "treatment" or "therapy" of a thrombotic disease in a mammal may include one or more of the following:

(1) arrest the development of thrombotic disease,

(2) prevent the spread of thrombotic disease,

(3) relieves thrombotic disease,

(4) prevent recurrence of thrombotic disease, and

(5) Palliating the symptoms of thrombotic disease

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant, in addition to the active ingredient, wherein the adjuvant includes excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, Lubricants, flavors and the like can be used.

In addition, the pharmaceutical composition (pharmaceutical composition) may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.

Formulation forms of the pharmaceutical composition (pharmaceutical composition) may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

The pharmaceutical composition of the present invention may be formulated in combination form by combining each drug alone or by mixing each drug. It is formulated and administered in a unit dosage form suitable for oral administration according to a conventional method in the pharmaceutical field. Formulations for oral administration suitable for this purpose include hard and soft capsules, tablets, dragees, film coated tablets, uncoated tablets, enteric tablets, powders, suspensions, syrups and the like. Such oral preparations may include, in addition to two pharmaceutically active ingredients, one or more pharmaceutically inert conventional carriers such as excipients such as starch, lactose, carboxymethylcellulose, kaolin, water, gelatin, alcohols, glucose. Additional ingredients may be included such as binders of oz, gum arabic, tragacanta rubber, disintegrants such as starch, dextrin, alginate, and lubricants such as talc, stearic acid, magnesium stearate, liquid paraffin. The pharmaceutical composition of the present invention may also add dissolution aids and the like for dissolution.

In addition, the pharmaceutical composition of the present invention can be administered parenterally, parenteral administration is not limited to the injection method such as subcutaneous injection, intravascular (vein and arterial) injection, intramuscular injection or intrathoracic injection. To formulate into parenteral formulations, thrombolytics and anti-inflammatory agents can be prepared in solution or suspension by mixing in water with stabilizers or buffers.

In one embodiment of the present invention, the active ingredient of the present invention may be included in 0.1 to 95% by weight relative to the total weight of the composition.

The pharmaceutical composition (pharmaceutical composition) of the present invention may contain the active ingredient in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier.

In the present invention, the "pharmaceutically effective amount" refers to an amount of an active ingredient that exhibits an alleviating, suppressing, improving and / or curative effect on a disease to be treated. The dosage of the active ingredient of the present invention varies in the range depending on the weight, age, sex, health condition, diet, administration time, administration method and severity of the disease of the patient. For example, therapeutically effective dosages can be initially determined using in vitro assays through cell culture. It will be possible to determine the amount effective for treatment without undue experimentation in the art, and this information can be used to more accurately determine the useful dose in humans.

The present invention also provides the use of a composition comprising the above thrombolytic and anti-inflammatory agents as an active ingredient for the manufacture of a medicament for the prevention or treatment of thrombotic diseases. The composition of the present invention comprising a thrombolytic and anti-inflammatory agent as an active ingredient can be used for the manufacture of a medicament for the prevention or treatment of thrombosis disease.

The present invention also provides a method for preventing or treating thrombosis disease, comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition (pharmaceutical composition) of the present invention.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “therapeutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, which Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example>

Preparation of Experimental Materials

Natokinase was purchased from WAKO (Richmond, USA). The activity of Wako nattokinase (360 IU / g) was accurately measured using a synthetic amido substrate (S-2222) that is specifically degraded by nattokinase. t-PA (Actilyse ^® ) was purchased from Boehringer Ingelheim (Mannheim, Germany) and Texamethason was purchased from Ilsung Pharmaceuticals (Seoul, Korea). Natokinase, t-PA and texametasone were dissolved in physiological saline solution and injected intravenously in rats.

Preparation of Laboratory Animals

Seven-week-old male Sprague-Dawley rats (200-220 g body weight) were obtained from Daehan-Biolink (Eumseong, Korea) and tested for 1 week in the laboratory environment. Animals were given free feed and water under constant environmental conditions (temperature 22 ± 2 ° C, relative humidity 40-70%, 12-hour contrast cycle, brightness 150-300 lux). All animal experiments were performed according to the standard working procedure of Chungbuk National University Laboratory Animal Research Center with the approval of Chungbuk National University Animal Ethics Committee.

Statistical analysis

Test results were expressed as mean ± standard deviation, and statistical significance of all results was one-way analysis of variance following Dunnett's multi-range test calibration using SPSS version 12.0 (SPSS, Chicago, USA). Analyze by. When P value <0.05, it was judged to be statistically significant.

<Example 1>

Antithrombotic Effect Assessment

In this experiment, the antithrombotic effects of nattokinase and t-PA (tissue-type plasminogen activator) alone and in combination with dexamethasone were evaluated.

Rats (n = 8 per group) were anesthetized by intramuscular injection of Zoletil (Virbac, Carros cedex, France; 1 mL / kg). Body temperature was maintained at 36-37 ° C. using a heat pad, the right carotid artery was exposed and separated from the vagus nerve and surrounding tissue. Aortic blood flow rates were monitored using a laser Doppler flowmeter (AD Instruments, Colorado Springs, USA). Arterial thrombosis was observed in Wattman No. moistened with 25% FeCl ₃ solution near the flowmeter probe. Induced by wrapping the artery for 10 minutes with 1 filter paper. After 5 minutes, thrombus-induced rats were dosed with nattokinase (25-400 mg / kg) and t-PA (5-30 mg / kg) alone, or with dexamethasone (2 mg / kg) intravenously Dosing and blood flow was monitored for 40 minutes. The drug administration method was injected intravenously. The rat was sacrificed under anesthesia and the visible thrombus artery was cut out. The arteries cut for tissue staining were fixed in 4% paraformaldehyde, and paraffin-embedded sections were stained with hematoxylin / eosin and analyzed by light microscopy.

As a result, as shown in FIG. 1A, when the 25% FeCl ₃ was applied to the outer surface of the carotid artery for 10 minutes, the blood flow decreased, and the blood flow stopped almost completely within 40-50 minutes. However, in the experimental group administered nattokinase intravenously 5 minutes after FeCl ₃ administration, it was confirmed that blood flow was preserved by inhibiting blood clot formation depending on the dose of nattokinase. In particular, it was confirmed that blood flow reduction was completely suppressed in the experimental group administered 75 or more mg / kg of nattokinase (○: solvent PBS group, ▽: 25 mg / kg nattokinase group, □: 50 mg / kg nattokinase group, ◇: 75 mg / kg nattokinase administration group, Δ: 100 mg / kg nattokinase administration group).

In addition, as shown in FIG. 1B, when th-PA was administered intravenously 5 minutes after the administration of FeCl ₃ , the thrombus formation was inhibited in a dose-dependent manner, thereby almost completely inhibiting the thrombus formation at the 10 mg / kg dose. It confirmed that it became. The calculated maximum effective dose was about 8.5 mg / kg (○: solvent PBS group, ▽: 5 mg / kg t-PA group, □: 7.5 mg / kg t-PA group, ◇: 10 mg / kg t-PA Administration group).

On the other hand, as shown in Figure 2a and 2b, the experimental group administered in combination with dexamethasone and nattokinase, compared to the experimental group administered with nattokinase alone, the blood flow improvement effect of arterial thrombosis was shown to be more excellent, such a combined use It was possible to effectively lower the effective dose of nattokinase (○: solvent PBS administration group, ●: dexamethasone (2 mg / kg) alone, ▽: nattokinase (25 mg / kg) alone, ▼: nattokinase (25) mg / kg) and dexamethasone (2 mg / kg) combination group, □: nattokinase (50 mg / kg) alone group, ■: nattokinase (50 mg / kg) and dexamethasone (2 mg / kg) combination group).

In contrast, as shown in FIG. 2B, the experimental group administered with dexamethasone and t-PA in combination with the t-PA alone did not significantly affect the blood flow improving effect of arterial thrombosis ( ○: solvent PBS group, ●: dexamethasone (2 mg / kg) alone group, ▽: t-PA (5 mg / kg) alone group, ▼: t-PA (5 mg / kg) and dexamethasone (2 mg / kg) ) Combination group, □: t-PA (7.5 mg / kg) alone group, ■: t-PA (7.5 mg / kg) and dexamethasone (2 mg / kg) combination group).

Through the above results, dexamethasone showed an excellent synergistic effect in improving arterial thrombosis blood flow when used in combination with nattokinase, compared to the synergistic effect of improving blood flow when used in combination with t-PA. Could.

In addition, as a result of observing the degree of arterial blockage caused by blood clots in tissues according to drug administration (intravenous injection) in an animal model of arterial thrombosis, as shown in FIG. 3, PBS or dexamethasone after induction of arterial thrombosis by applying FeCl ₃ When only (2 mg / kg) was administered, it was confirmed that the artery was completely blocked by the thrombus at 40 minutes. On the other hand, in the experimental group co-administered with nattokinase (25 mg / kg) or t-PA (5 mg / kg), the arterial lumen was not completely blocked and the thrombus became small and loose.

<Example 2>

Hemorrhagic Side Effects Assessment

In this experiment, we assessed the degree of bleeding in the tissue following drug administration (intravenous injection) to evaluate the hemorrhagic side effects of administration of nattokinase and t-PA alone or dexamethasone in combination with animal models that induced arterial thrombosis. Observation was performed through tissue staining.

In Example 1, the rats to which the drug was administered after inducing arterial thrombosis were sacrificed, and the sacrificed rats were perfused through intraventricular injection of phosphate buffered saline. All organs were dissected to examine the hemorrhagic findings on the surface. Paraffin embedded sections were stained with hematoxylin-eosin and observed under an optical microscope.

As a result, it was confirmed that bleeding (arrow) was seen in the lungs (A) and thymus (B) in the experimental group administered at 300 mg / kg dose of nattokinase (see FIG. 4). In addition, in the case of t-PA, hemorrhagic lesions were increased in both lungs (A) and thymus (B) depending on the dose. Particularly, hemorrhagic lesions were observed in the experimental group administered with a low dose of 10 mg / kg. (See FIG. 5).

Meanwhile, the degree of blood flow improvement, the degree of bleeding, and the safety margin according to the administration of nattokinase and t-PA alone or in combination with dexamethasone in the arterial thrombosis animal model are shown in Table 1 below.

Blood flow improvement effect, bleeding degree and safety margin by administration of nattokinase and t-PA alone or in combination with dexamethasone in animal models of arterial thrombosis process Dose (mg / kg) Blood flow improvement effect (%) Bleeding score Safety margin Nattokinase 25 59 - 4.0 50 86 - 75 94 - 100 96 - 200 100 - 300 100 + Dexamethasone
+ Nattokinase 25 75 - 8.0 50 94 - 300 100 - 400 100 + t-PA 5 63 - 1.2 7.5 76 - 10 100 + 20 100 + 30 100 ++ Dexamethasone
+ t-PA 5 68 - 2.4 7.5 78 - 10 100 - 20 100 +

1)-: normal, +: <5% bleeding, ++: <10% bleeding; +++: <50% bleeding; ++++:> 50% bleeding.

2) blood flow improvement: blood flow rate maintained for 40 minutes

3) Determination of bleeding score: Pathologic clinical findings based on bleeding range (maximum ++++)

4) safety margin =

* Effective effective dose (ED ₉₀ ): A dose that maintains blood flow more than 90% for 40 minutes

5) In the above table, the dose means the treatment dose of nattokinase or t-PA, and dexamethasone treated in combination with this is treated with a 2 mg / kg dose.

As a result, it was confirmed that the bleeding response to thrombolytic agents such as nattokinase and t-PA was attenuated by the combined use of dexamethasone (2 mg / kg), and the dose at which the bleeding reaction of nattokinase and t-PA began to occur. Increased to 400 mg / kg and 20 mg / kg, respectively. In other words, the bleeding reaction occurred from 300 mg / kg and 10 mg / kg when the nattokinase or t-PA alone was administered without the simultaneous administration of dexamethasone, but 400 mg / kg and the dexamethasone, respectively. It was confirmed that the bleeding reaction occurs from 20 mg / kg.

In particular, the standard safety margins based on the ratio of the blood flow improvement effect (effective dose) to the toxic (bleeding) doses of thrombolytic agent nattokinase and t-PA were estimated to be 4.0 and 1.2, respectively. Improving the safety of nattokinase and t-PA doubled the safety margin to 8.0 and 2.4, respectively.

Therefore, the above results show that the combined use of dexamethasone effectively reduces the bleeding side effects of thrombolytic agents such as nattokinase and t-PA.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (10)

As a pharmaceutical composition for thrombolytic aid,
The composition includes dexamethasone as an active ingredient,
The thrombolytic agent is a nattokinase or a tissue-type plasminogen activator (tissue-type plasminogen activator) thrombolytic aid pharmaceutical composition. delete delete The method of claim 1,
The composition is a thrombolytic supplemental pharmaceutical composition, characterized in that to increase the thrombolytic activity of the thrombolytic agent and to reduce the bleeding side effects of using the thrombolytic agent. Thrombolytic nattokinase or tissue-type plasminogen activator; And pharmaceutical composition for inhibiting platelet aggregation or thrombolysis comprising dexamethasone as an active ingredient. delete delete The method of claim 5,
The dexamethasone (dexamethasone) is a pharmaceutical composition, characterized in that to increase the thrombolytic activity of thrombolytics and to reduce bleeding side effects of the use of thrombolytics. A pharmaceutical preparation for inhibiting platelet aggregation or thrombolytic activity prepared from the composition of claim 5 or 8. The method of claim 9,
The formulation is selected from the group consisting of hard and soft capsules, tablets, dragees, film coated tablets, uncoated tablets, enteric tablets, powders, suspensions, syrups and injections.

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