WO2012102555A2 - Fucoidan for thrombolysis, derived from seaweed - Google Patents

Abstract

To study different types of thrombolytic agents, both thrombolysis and stimulation of fucoidan were studied in a mouse model of ferric chloride-induced arterial thrombosis and were compared with those of a heparin and tissue plasminogen activator (t-PA). Thrombosis was induced by applying a filter paper saturated with 5% FeCl₃ to the left carotid artery. 20 minutes after complete occlusion, several test preparations including a fucoidan source from Undaria pinnatifida sporophylls were intravenously injected to each mouse group by various dosages of 10-200 mg/kg^-1. To study the stimulation effect of fucoidan with respect to t-PA, fucoidan added in a predetermined amount of t-PA was injected by a dose of 1-10 mg/kg. Then, the time to reperfusion was measured. As a result, an occluded aorta was dose-dependently reperfused, and the reperfusion was observed at 37.5±12.4 minutes after the administration of 100 mg/kg^-1 of unfractionated fucoidan. The time to reperfusion was retarded to 55.0±8.0 minutes when a large amount of fucoidan such as 1 g/kg^-1 was administered to a mouse provided with low molecular weight fucoidan (LMWF). Reperfusion occurred at 16.67 minutes after administration in a mouse of a control group provided with 20 mg/kg^-1 of t-PA. On the contrary, reperfusion was not observed in an occluded mouse provided with heparin (p<0.01). The occluded artery was recovered without reperfusion at 17.2 minutes after injection if the least amount of fucoidan added in a predetermined amount of t-PA was provided. All the data show that fucoidan can serve as both a thrombolytic agent and a stimulant with respect to thrombolysis activation of t-PA in a thrombosis model, differently from heparin.

Description

 【Specification]

 [Name of invention].

 Fucoidan for thrombolysis from algae

 Technical Field

 The present invention relates to fucoidan for thrombolytic fucoidan derived from algae, and more particularly to fucoidan having a thrombolytic use characterized by branched structures derived from seaweed such as seaweed spores and rhubarb and not fractionated. will be.

 Background

 Eurokinase or tissue plasminogen activator (t-PA) is a thrombotic agent for the treatment of severe or widespread deep vein thrombosis, pulmonary embolism, myocardial infarction, and treatment of occluded intravenous or dialysis cannula. Used as an enemy. Hemolytic therapy with recombinant t-PA is effective in acute myocardial infarction, but this treatment is limited by the significantly slower reperfusion rate and frequent early reclosure. Coronary artery reobstruction after thrombolytic therapy occurs in one third of patients despite heparin administration. Heparin promotes the binding of thrombin to fibrin polymers by forming ternary complexes, thus limiting the preventive effect of coronary re-obstruction. Early reclosure may be due to a mechanism by which thrombin is released from thrombi during thrombolytic therapy. The active form of thrombin (CPU) has recently been known to activate procarboxypeptidase U, which downregulates endogenous fibrin lysis. Therefore, one way to improve thrombolytic efficacy is to theoretically bind t-PA to low-molecular-weight thrombin direct inhibitors that may have a pro-thrombolytic effect, thereby inhibiting fibrin-bound thrombin and / or CPU It may be to inhibit activation.

Coronary thrombosis is usually caused by rupture of atherosclerotic plaques. Arterial thrombus is often rich in platelets because of the local development of strong platelet agonists such as trumbin and the high shear forces present in diseased arteries. However, platelet-rich thrombi are poorly dissolved by t-PA. Therefore, research continues on current thrombolytics and other types of thrombolytics that maximize the efficacy without the risk of additional bleeding. Fucoidan is known to have a stimulatory effect on t-PA induced thrombolytic activity in most in vitro studies, which protects plasmin activity from α ₂ -plasmin inhibitors and speeds up fibrin polymer formation. It suggests a mechanism for reducing The magnitude of the stimulatory activity depends on the degree of sulphation and is increased by additional chemical sulphation of fucoidan. Fucoidan, a branched sulfated fucan extracted from brown algae, has anti-ungal and antithrombotic effects mediated by direct thrombin inhibition, whereas linear fucans derived from dermal animals have been reported for mammalian glycosaminoglycans. As indicated, thrombin inhibition requires the presence of heparin cofactor ( _co f _ac tor) II. Since thrombin promotes thrombus formation by converting fibrinogen into fibrin (protrumbo) and promotes thrombus stabilization by converting proCPU into CPU (antifibrin soluble), this direct thrombin inhibition may facilitate the endogenous fibrin dissolution system. have. Anticoagulation and antithrombotic activation of fucoidan have been widely studied, but little is known about the in vitro fibrin lysis system. Soeda et al. Reported that high sulfated fucoidans prepared by chemically sulphating commercially available material (Fucus. Vesiculosus) increase the t-PA-catalyzed activation of Lys-plasminogen and from α ₂ -plasmin inhibitors. T- by protecting plasmin activity

It has been reported to stimulate PA-induced plasma clot lysis. Doctor et al. Demonstrated that purified commercial fucoidan promotes the activation of Glu- and Lys-plasminogen by two—chain t-PA and low molecular weight u-PA. Fucoidan-Sepharose affinity chromatography has also been shown to bind Fucoidan to PAI-Kplasminogen-activator inhibitor-1 and reduce PAI-1 activation. Nishino et al. Also showed that E.Kurome-derived fucoidan significantly increased plasmin generation by HMW u-PA-mediated plasminogen activation in addition to t_PA-mediated plasminogen activation. All these in vitro prior art suggest a role of fucoidan in thrombolytic activity, but no reports have been made of fucoidan alone to have a thrombolytic effect in an in vivo thrombosis model.

 Republic of Korea Patent Application No. 10-2004-0003912 'Cardiovascular disease improvement composition' contains 2 to 70% by weight of alginic acid derived from seaweed, 0.5 to 30% by weight of fucoidan, 1 to 50% by weight of laminarin, phloroglucinol 2 A cardiovascular disease improving composition is disclosed, comprising 70% by weight to 0.01% by weight of fucoste, and

 Korean Patent Application No. 10-2008-0120587 'Pharmaceutical composition for promoting bone formation and active containing fucoidan' describes a pharmaceutical composition for promoting bone formation and active containing fucoidan as an active ingredient,

Republic of Korea Patent Application No. 10-2006-0009097 'An anti-obesity gochujang containing fucoidan' describes the manufacturing method of anti-cancer functional enhancement kochujang characterized by adding 0.5-3% by weight of fucoidan powder during the preparation of kochujang However, the use of Fucoidan as a thrombolytic agent in any of the prior arts has been described. There is no.

 [Detailed Description of the Invention]

 [Technical problem]

 In the present invention, the thrombolytic effect of unfractionated fucoidan derived from seaweed spores (Undaria pinnatifida sporophylls) was studied in a thrombosis model mouse, and the commercial fucoidan source derived from heparin, Fucus vesiculous, t_PA and Compared to urokinase, it is an object to provide fucoidan with excellent thrombolytic and t-PA stimulatory effects.

 Technical Solution

 <ιο> In the present invention, seaweed, in particular seaweed spores (Undaria pinnafida sporophylls: UPS-

UF) and fucoidan for thrombolysis characterized by a branched structure that is not fractionated from rhubarb.

 In addition, the present invention provides a fucoidan for t-PA stimulation characterized by branched structures derived from algae, in particular seaweed spores (UPS-UF) and rhubarb, which are not fractionated.

According to the present invention, thrombolysis is also observed using fucoidan alone in an in vivo thrombosis model. This is in contrast to the antithrombotic and anticoagulant properties as heparin sulphated polysaccharides, which do not show a thrombolytic effect alone.

 This indicates that the mechanism of action of the thrombosis system and fucoidan is completely different from the mechanisms supporting the stimulatory activity. Fucoidan is known to have binding affinity with both thrombin and PAI-1, and thus can act as an inhibitor of them.

. have.

 Like plasminogen, t-PA is a lysine exposed on partially degraded fibrin

It has a high affinity for lysine.

As can be seen from FIG. 3, locally released and circulating t-PA converts plasminogen into plasmin, which in turn decomposes fibrin, thus dissolving its dissolution. Plasminogen activator inhibitors (PAI-1) are the major inhibitors of t-PA in vascular components. Thrombin activates more platelets to mobilize to the site of injury, and then develops fibrin stratified for stabilization of the primary platelet plug. Thrombin and FXa, which are formed during the coagulation process, are effective "causes" of t-PA release. Thrombin converts fibrinogen into fibrin (prothrombo) to promote thrombus formation and by converting proCPU to CPU (antifibrin soluble). As it promotes thrombus stabilization, the combination of fucoidan and thrombin may increase t-PA release or endogenous fibrin The dissolution system can be facilitated. In addition, the binding of fucoidan and PAI-1 can reduce the inhibitory activity of PAI to t-PA and effectively facilitate thrombolysis.

 <16> Polymorphonuclear neutrophils (PMN) in plasma accumulate in the blood clots during arterial thrombosis, and after the formation of thrombus, especially in the case of Fucoidan injection, Plia is distributed not only in the thrombus but also in the thrombogenic arterial endothelial tissue. -PA can be induced or directly activated to induce thrombolysis by releasing biplasmin protease in PMN.

 When t-PA binds to fibrin, the rate at which plasminogen is activated by plasmin decreases.

 Increases over 100¾. Thus, plasmin generation is a sequential and ordered activation mechanism involving the formation of three-way complexes between fibrin, plasminogen and t-PA. Therefore, the combination of fucoidan and PAI-1 resulted in a rapid and substantial increase in local intraarterial t-PA concentrations within a large intracellular storage pool of t—PA due to a regulated pathway. The binding of and fibrin may be facilitated, thus facilitating plasminogen activation (FIG. 3). It may also increase the production of plasminogen or endogenous t-PA from endothelial cells, resulting in increased plasmin concentrations and thrombolysis in arteries occluded by blood clots.

 As a direct inhibitor of thrombin, fucoidan reduces t- by lowering proCPU activation.

The thrombolytic rate of PA can be increased more efficiently, and the plasma nogen-fibrin binding rate can be increased as shown in FIG. 3. In other words, the binding of fucoidan and PAI-1 or thrombin can facilitate the thrombolytic system in the occluded artery by effectively increasing vascular plasmin.

LMWF fractions retarded thrombolysis in the occlusion artery until injected at a dose of 1000 mg kg ^"1, 10 times more than unfractionated fucoidan. In addition, groups of mice receiving Fucus fucoidan were treated with seaweed. Reperfusion at 200 mg kg-1 was nearly doubled in time-to-reper fusion compared to mice receiving spore fucoidan, therefore the thrombolytic activity of fucoidan was determined by sulfated fucoidan. It may depend on both the degree and chemical structure Other prior arts describe mechanisms that support fucoidan's thrombolytic activity in terms of stimulating the endogenous release of t-PA and / or PAI-1 potential inhibitors. It is necessary to

<20> A minimum amount of fucoidan, K g kg-, in a constant t-PA dose without reperfusion.

Recovery of the occlusion artery was not observed with an additional injection of 1 UPS-UF. Such No irritant effect is observed at 5 mg kg-1 and therefore appears to be dose dependent. This finding is consistent with the content of the prior art for in vitro studies.

More interestingly, blood flow recovery in fucoidan-stimulated reperfusion mice was not reoccluded compared to the heparin-treated group administered at the same dose (10 mg kg-1) (p <0.05). Active thrombin may be released from thrombi during thrombolytic therapy. Aspect differences between fucoidan and heparin may be due to differences in activity with thrombin. Heparin promotes the binding of thrombin and fibrin polymers to assist in occlusion, while fucoidan can bind directly to thrombin to inhibit fibrin formation, as shown by the mechanism of FIG. 3.

 Carotid artery clots are rich in platelets and contain high concentrations of PAI-1. Therefore, carotid artery thrombi is poorly dissolved by t-PA, mainly due to PAI-1. Since algae fucoidan inhibits platelet masses and is a possible inhibitor of PAI-1, it can improve the thrombolytic dissolution of t-PA.

 The thrombolysis of the occlusion artery was observed for the first time in an in vivo thrombosis model by direct administration of seaweed fucoidan (from Undaria pinnatifida sporophylls) alone. Fucoidan sources obtained from different seaweed species showed slow reperfusion at higher doses. This is in contrast to heparin, which did not exhibit this effect. In addition, when the t-PA is injected, the fucoidan exhibits a thrombolytic effect at a minimum dosage that is insufficient to exhibit thrombolysis, and exhibits a synergistic anti-stimulatory effect. No occlusion was observed in all of the fucoidan-mediated reperfusion arteries of occlusion mice. Therefore, the data indicate that fucoidan can act as both a thrombolytic and synergistic anti-stimulator for t-PA thrombolytic activity in a thrombosis model. This activity of the fucoidan may be due to its binding properties with thrombin or PAI-1. Advantageous Effects

 According to the present invention, fucoidan derived from seaweeds such as seaweed spore (Undaria pinnatifida sporophyl Is MJPS-UF) and rhubarb has a synergistic anti-stimulatory effect that adds a t-PA effect, and even when used alone, a thrombolytic effect Has the effect of indicating. [Brief Description of Drawings]

FIG. 25 is an image showing induction of arterial thrombosis by applying 5% ferric chloride for 3 minutes on the left carotid artery area of Balb / c mouse, and FIG. Lb shows the intravascular space filled with thrombus. A cross-sectional view of a blood-facing directional thrombosis region, and FIG. lc is a cross-sectional view of an intermediate thrombosis region showing an intravascular space filled with thrombi. here Fucoidan 60 to 200 mg kg—1 was injected by tail vein down 20 minutes after complete occlusion.

 FIG. 2A is an image showing the ventral neck view through an anatomical microscope (mouse head up), and FIG. 2B is a cross-sectional view of the reperfusion region where blood flow has been restored. Figure 2C is a cross-sectional view of the blood-facing directional thrombosis region showing the vascular space in the process of polyclonal neutrophils are distributed on the endothelial tissue when the fucoidan is administered and thrombolysis is dissolved, Figure 2D It is a cross-sectional view of the middle region of thrombosis showing space.

 FIG. 3 is a schematic diagram illustrating the mechanism of action of fucoidan in thiolytic activity.

 [Mode for Carrying Out the Invention]

 <28> Materials and Methods

 <29> substances

Unfractionated fucoidan from seaweed spores (Undaria pinnatifida sporophylls: UPS-UF) was prepared. That is, fucoidan was continuously extracted three times at 65 ^° C. for 1 hour with a hydrochloric acid solution having a pH of 2.0, and the filtrates were collected by filtration and concentrated, and then ethanol three times was added to obtain raw fucoidan. Acid fractions were added to the raw fucoidan solution, which was re-dissolved in distilled water, for the partial purification to precipitate acidic fractions, and then fractionated by the addition of calcium chloride. The remaining alginate was separated using ethanol in the solution obtained after dialysis. Purification was by using precipitation. Additional purification was performed using XAD hydrophobic gel chromatography to remove polyphenol components. Low molecular weight fucoidans (UPS-LMWF, 6-10 kD) were produced by radical depolymerization process. Another unfractionated fucoidan source from Fucus vesiculosus (FV-UF) was purchased from Sigma (Sigma, St. Louis, MO) and used after further purification using hydrophobic gel continuous chromatography. Sanofi Pharmaceuticals (Sanofi)

Heparin from Pharmaceuticals, Seoul, Korea. t-PA was provided by Boeringer Ingelheim.

<31> arterial thrombosis model

<32> In this study, Bal / c mice (18-25 g, Tacoma, Mass.) Were used. Animals were kept in cages and managed according to the Guide for the Care and Use of Laboratory Animals. Procedures for the use of laboratory animals can be found in the Experimental Animal Steering Committee Institutional Animal Care and Use Co 隱 it tee. lOOg per body weight. Mice were anesthetized by intraperitoneal administration of 7.5 mg of ketamine hydrochloride (Hoffmann-La Roche, Swiss Bazell) and 2.5 mg of xylazine. After anesthesia, the skin just above the area of the right carotid artery was dissected. Subsequently, the fascia was blunt dissected and exposed the left part of the carotid artery. Miniature Doppler Flow Probes

Carotid artery blood flow was measured using a miniature Doppler flow probe (Model 0.5 VB, Transonic System, Ithaca, NY). Two filter papers saturated with 5% ferric chloride (Sigma, St. Louis, MO) (1 X 2 2) (Gel Blot Paper, GB003, Schleicher and Schuell, Keene, New Hampshire, USA ) Was applied to induce thrombosis. The filter paper was placed (one below and one above) on the opposite side of the carotid artery in contact with the vascular envelope membrane surface. The filter paper was removed after 3 minutes of application. Carotid blood flow was observed every 2 minutes after applying the filter paper.

 <33> Thrombolysis Treatment (Treatment)

Mice (6-8 weeks old, 20-25 g weight) were used as carotid artery injury models. Filter paper saturated with 5% FeCl ₃ on the arterial surface immediately adjacent to the Doppler flow rate probe (1 X

2v) was released for 3.0 minutes to induce carotid artery injury (FIG. 2A). Conical ultrasonic gels were applied to the surface of the flow rate probe to ensure efficient contact with the artery. The time to occlusion, defined as the Doppler beat, disappeared. 20 minutes after occlusion, UF, LMWF, unfractionated porcine heparin, urokinase and recombinant tissue plasminogen activator (t-PA) were injected intravenously via the tail vein into a 10-1000 mg / kg dose. The time to reperfusion, which is defined as the Doppler bit disappears, was recorded. Carotid blood flow was observed for 60 minutes after reperfusion or 1.5 hours after drug infusion if no reperfusion occurred.

To study the stimulating effect of fucoidan on the thrombolytic activity of t-PA as a direct inhibitor of thrombin, 1 to 10 mg kg— ¹ in addition to 15 mg kg ^“1 or 10 mg kg ^{“ 1} of t-PA. Doses of unfractionated fucoidan or heparin were injected. The time-reperfusion change was measured.

<36> Statistical Analysis

Data is expressed as mean: t SEM. The thrombolytic activity of the animals in group 5 was compared using the unbound Student's t test. Values with p <0.05 were considered significantly different. Data was expressed by mean SEM. ratio Groups were compared using the binding Student's t test.

<38> results

<39> the development of thrombosis

<40> FeCl ₃ (concentrations of 2 to 10¾) on carotid thrombosis formation-dependent effects

Proven in Balb / C mice. Thrombosis formation and complete occlusion can be seen by decreasing or disappearing the Doppler bit in the Doppler flow rate probe. No effect on blood flow was observed after 2% FeCl ₃ treatment. At 5% FeCl ₃ concentrations, all five cases of 5 occlusions were observed within 10 minutes as shown in FIG. Excisions of arterial thrombi were stained with H / E and then examined under an optical microscope, and the vascular space was completely filled with thrombi as shown in FIG.

<41> thrombolytic activity

To demonstrate its usefulness in the evaluation of thrombolytic drugs, the effects of fucoidan and heparin were studied in comparison with t-PA and urokinase using a 5% FeCl ₃ -induced arterial thrombosis model. The results are summarized in Table 1 below.

<43> [Table 1]

Thrombolytic Effects of Intravenously Injected Fucoidan, Heparin, tPA, and uPA on a FeCl ₃ -Induced Arterial Thrombosis Mouse Model

<45> 폐색성 혈전이 형성된 후에, 각 그룹의 5마리 마우스는 60 내지 200 mg kg 투약량의 UPS— UF, UPS-LMWF, FV-UF， 헤파린을 제공받았고， 한 그룹의 5마리 마우스 는 60 내지 1000 mg kg^'1 투약량의 UPS-LMWF를 제공받았으며， 2개 그룹의 5마리 마 우스는 대조군으로서 10 내지 100 mg kg^"1 투약량의 t-PA 및 유로키나아제를 제공받 았고， 폐색 동맥의 재개방이 일어나는 재관류 투약량을 연구하였다. t-PA 처리된 마우스 5마리 중 5마리에서는 경동맥이 15 내지 30분 이내에 (평균값， 16.67 土 2.88분) 복구되어, 최대 혈류 약 100%에서 혈관 개방을 유지하는데 있어서 완전한 효능을 나타낸 반면, 유로키나아제-처리된 마우스 5마리 중 5마리에서는 혈류 관측 2시간동안 재관류가 발생하지 않았다. 100 mg kg-1의 UPS-UF를 투여한지 30 내지 60분 이내에 (평균값， 37.5士 12.4분) 재관류가 관찰되었다. 이러한 재관류 투약량 은 UPS-LM 을 주입하는 경우에는 1000 mg kg-1까지 증가하였다. FV-UF를 100 mg kg^"1 투약량으로 주입할 때， 투약량이 200 mg kg-1으로 증가할 때까지는 재관류가 관찰되지 않았고， 이어서 투여한자 63.3土 7.2분후에 재관류가 발생하였다. 복원 된 동맥 절제부의 현미경 이미지는 도 2b에 나타난 바와 같이 혈류로 인한 흔량의 적혈구 세포를 나타낸다. 1시간 이내에는 모든 재관류된 마우스에서 재폐색이 관 찰되지 않았다. 이와 대조적으로， 헤파린 60 내지 1000 mg kg¹ 투약량으로 처리한 5마리 마우스중 5마리에서는 혈류를 관측하는 1.5사간동안 재관류가 발생하지 않았 다-

After the obstructive thrombus was formed, 5 mice in each group received 60-200 mg kg doses of UPS—UF, UPS-LMWF, FV-UF, heparin, and 5 mice in a group received 60-60 mg. 1000 mg kg ^'1 dose of UPS-LMWF was received, two mice in two groups received 10-100 mg kg ^"1 dose of t-PA and urokinase as controls, and reopening of the occlusion artery The resulting reperfusion dose was studied: In 5 of 5 t-PA treated mice, the carotid artery recovered within 15 to 30 minutes (mean value, 16.67 土 2.88 minutes), which was complete in maintaining blood vessel opening at about 100% of the maximum blood flow. On the other hand, five out of five eurokinase-treated mice did not experience reperfusion for two hours of blood flow observations within 30 to 60 minutes of administration of 100 mg kg-1 UPS-UF (mean value, 37.5 mbar). 12.4 minutes) Perfusion tube This reperfusion dose increased to 1000 mg kg-1 when injecting UPS-LM. When FV-UF was injected at a 100 mg kg ^"1 dose, the dose increased to 200 mg kg-1. Reperfusion was not observed until then, and reperfusion occurred after 7.2 minutes of administration. Microscopic images of the reconstructed arterial ablation show common red blood cells due to blood flow, as shown in FIG. 2B. Reclosure was not observed in all reperfused mice within 1 hour. In contrast, five of the five mice treated with a dose of 60 to 1000 mg kg ^{1 of} heparin did not experience reperfusion during 1.5 hours of blood flow monitoring—

<46> Effects of Fucoidan on t-PA-induced Thrombolysis

 Time—reperfusion was measured as a function of fucoidan infusion dose in addition to the t-PA dose, and the results are summarized in Table 2 below.

 <48> [Table 2]

<49> Synergistic Antistimulatory Effect of Fucoidan on Thrombolytic Activity of t-PA

 Synergistic anti-stimulatory effect of fucoidan on thrombolytic activity of t-PA In mice given 10 mg kg-1 of t-PA, no reperfusion was observed until 60 minutes post-injection, followed by an additional 10 mg of unfractionated fucoidan Intravenous injection and reperfusion occurred 17.2 minutes after fucoidan injection. In contrast, reperfusion was observed at 9.3 μs 3/7 minutes after injecting 10 mg kg-1 of heparin in addition to t-PA, followed by intermittent reobstruction after 5 minutes, and bleeding in adjacent injured arteries. As it continued, reperfusion was observed again. However, this reclosure was observed when injecting 1.25 mg kg-1 of heparin in addition to t-PA. In mice receiving 15 mg kg-1 of t-PA, reperfusion occurred 24 min 7.1 min after additional injection of fucoidan 5 or 10 mg kg-1, and the change in time-reperfusion was not statistically significant.

Claims

[Range of request]  [Claim 1]  Thrombolytic fucoidan derived from algae, including seaweed spores (Undaria pinafida sporophylls (UPS-UF) and rhubarb).  [Claim 2]  Fucoidan for algae thrombosis, characterized by branched and unfractionated branched structures of claim 1.  [Claim 3]  Fucoidan for seaweed thrombosis, characterized in that the low molecular weight structure of claim 2.  [Claim 4]  Fucoidan for synergistic anti-stimulation of tissue plasminogen activator (t-PA) derived from seaweeds including Undaria pinafida sporophyl Is (UPS-UF) and rhubarb.  [Claim 5]  The fucoidan for synapse anti-stimulation of tissues, plasminogen activator (t-PA), characterized by branched and unfractionated branched structure according to claim 4.  [Claim 6]  A fucoidan for tissue plasminogen activator (t-PA) synergistic anti-¾ stimulation, characterized by a low molecular weight structure.