DO3A-based thrombus inhibitor or hemolytic agent containing gadolinium complex

Abstract

The present invention has found that a DO3A-based substance containing a gadolinium complex, which is conventionally used as an anti-inflammatory agent, not only does not participate in a platelet function for stopping bleeding of an injured or wound site, but also has a novel effect of inhibiting thrombin-induced thrombus or dissolving blood. Disclosed is a possibility of application of a DO3A-based substance containing a gadolinium complex in a new use as a thrombus inhibitor or a blood coagulant.

Description

Technical Field

[0001] The present invention relates to a DO3A-based pharmaceutical composition containing a gadolinium complex, and more specifically, to an invention in which the pharmaceutical composition, which is known as an anti-inflammatory agent, has a new effect of inhibiting thrombus or dissolving blood. Background Art

[0002] Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX-1 and COX-2 enzymes involved in the synthesis of prostaglandins, and through this action produce analgesic, anti-inflammatory, antipyretic effects, etc. COX-1 activates prostaglandins and thromboxane A2, responsible for the protection of gastric mucosa, maintenance of renal function, platelet hemostasis and other defensive effects, COX-2 activates inflammatory mediators and cytokines in various tissues such as vascular endothelial cells, causing pain and inflammation.

[0003] According to the selectivity of these COX substances, NSAIDs are divided into non-selective NSAIDs and COX-2 selective NSAIDs. Since non-selective NSAIDs inhibit COX-1 together, they have a fatal weakness, which is to inhibit the protective effect of gastric mucosa, causing antiplatelet effect, resulting in frequent gastrointestinal side effects such as peptic ulcers. Subsequently, COX-2 selective NSAIDs appeared, which not only selectively inhibit COX-2, but also maintain the anti-inflammatory and analgesic effects, and are expected to solve the various side effects caused by blocking COX-1.

[0004] It is well known that COX-2 selective NSAIDs can reduce the incidence of gastrointestinal complications, but their cardiovascular and renal toxicity side effects are still controversial. When NSAIDs inhibit COX-2, the production of prostacyclin is inhibited, while the antiplatelet effect is reduced, causing sodium and water retention, and further promoting platelet coagulation through COX-1, increasing the risk of thrombosis and embolism, which will increase the occurrence of cardiovascular events.

[0005] In addition, diflunisal is known as a non-selective COX inhibitor or a weak COX-1 selective inhibitor. Referring to Korean Patent Nos. 10-2018-0033426 and 10-2020-0119574, it is disclosed that a compound of diflunisal bound to DO3A containing a Gd complex and a compound of diflunisal bound to DO3A are respectively used as selective COX-2 inhibitors and can be used as anti-inflammatory agents. According to the contents of the above two patents, it can be known that a compound of diflunisal bound to DO3A is used as an anti-inflammatory agent as a selective COX-2 inhibitor, and can be used as an anti-inflammatory agent for brain inflammatory diseases. However, from the contents of Korean Patent No. 10-2020-0119574, although the effect as an anti-inflammatory agent is proved, the effect of thrombus inhibition is not disclosed. Summary of the invention

[0006] One object of the present invention is to provide a pharmaceutical composition that has been developed as an anti-inflammatory agent and has the effect of a new pharmaceutical use as a thrombus inhibitor.

[0007] A thrombus inhibitor or hemolytic agent for achieving one purpose of the present invention comprises a compound having a structure represented by the following formula 1. The compound having the structure of the following formula 1 is a substance known to be used as an anti-inflammatory agent. In the prior art, cardiovascular side effects are solved by co-administering the above-mentioned compound with other thrombus inhibitors. However, the inventors of the present invention have found that the compound having the structure of the following formula 1 provides a new effect of inhibiting thrombus or dissolving blood in addition to the anti-inflammatory effect, and thus, the compound having the structure of the following formula 1 can be used as a new drug for thrombus inhibitors or hemolytic agents.

[0008] [Chemical formula 1]

[0009]

[0010] In Chemical Formula 1,

[0011] A represents *-(CH2) n -A 1 -*,

[0012] n represents any integer from 0 to 5,

[0013] A 1 Indicates *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-* or *-O-*,

[0014] Linker indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2-OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -* or *-L 1 -COO-L 2 -*,

[0015] L 1 represents a straight chain or branched (C1-C30) alkyl group,

[0016] L 2 represents a single bond or a straight chain or branched (C1-C30) alkyl group,

[0017] R 1 and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, R 2 represents a straight chain or branched (C1-C10) alkyl group,

[0018] B stands for Wherein, * indicates the binding site.

[0019] Preferably, the thrombus inhibitor or hemolytic agent of the present invention has a structure represented by the following formula 2, wherein diflunisal is combined with DO3A containing a gadolinium complex:

[0020] [Chemical formula 2]

[0021]

[0022] When blood vessels in the body are damaged and bleeding occurs, hemostasis is achieved through the primary hemostasis process and the secondary hemostasis process. Primary hemostasis is achieved through platelet hemostasis, that is, platelets adhere to and aggregate at the site of damaged blood vessels to achieve hemostasis, and secondary hemostasis is achieved through coagulation factor hemostasis, that is, through the activation of coagulation factors, hard fibrin is formed on the aggregated platelets to completely stop bleeding. During the primary hemostasis process, the shape of platelets changes from disc to sphere, and they adhere and aggregate (aggregation) on the collagen of the blood vessels. The adhered platelets are activated and release calcium (Ca 2+) and ADP (adenosinediphosphate) and other granular substances. At this time, the secreted ADP combines with the surrounding platelets, activates the platelets, and achieves platelet aggregation. Secondary hemostasis is to form hard fibrin by activating coagulation factors. The activation of coagulation factors produces thrombin through intrinsic and extrinsic pathways, and thrombin acts on fibrinogen to form fibrin polymers. Fibrin or fibrin polymers combine with the primary hemostatic plug formed by platelet adhesion and aggregation during the primary hemostasis process to form a more solid and hard secondary hemostatic plug, thereby achieving the purpose of complete hemostasis.

[0023] The thrombus inhibitor or hemolytic agent of the present invention is characterized by inhibiting platelet aggregation induced by thrombin, i.e., secondary hemostasis. The thrombus inhibitor or hemolytic agent of the present invention has the effect of inhibiting platelet aggregation induced by thrombin in the secondary hemostasis process, rather than inhibiting the induction of platelet adhesion to the damaged part in the primary hemostasis process.

[0024] In one embodiment, the thrombus inhibitor or hemolytic agent can control the activation of integrin αIIBβ3. Integrin αIIBβ3 is a protein involved in adhesion and mediates platelet aggregation and interaction with other adjacent tissues. The thrombus inhibitor or hemolytic agent of the present invention can inhibit the activation of integrin αIIBβ3 in a concentration-dependent manner. In the present invention, "concentration dependence" means that the degree of effect increases with increasing concentration, or the degree of effect decreases with decreasing concentration. In other words, as the concentration of the thrombus inhibitor or hemolytic agent of the present invention increases, the activation of integrin αIIBβ3 involved in platelet aggregation can be effectively inhibited.

[0025] In one embodiment, the thrombus inhibitor or hemolytic agent can inhibit the secretion of adenosine triphosphate (ATP). Adenosine triphosphate is a substance released when platelets are activated and can bind to the P2Y2 receptor on the surface of vascular endothelial cells. The present invention can affect platelet activation by inhibiting the secretion of ATP.

[0026] The anti-inflammatory agent for achieving other purposes of the present invention has a structure represented by the following formula 1 and has the property of inhibiting thrombus or dissolving blood. Since the anti-inflammatory agent has the property of inhibiting thrombus or dissolving blood, it has the advantage of not needing to be administered together with other drugs that inhibit thrombus.

[0027] [Chemical formula 1]

[0028]

[0029] In the chemical formula 1,

[0030] A represents *-(CH2) n -A 1 -*,

[0031] n represents any integer from 0 to 5,

[0032] A 1 Indicates *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-* or *-O-*,

[0033] Linker indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2 -OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -* or *-L 1 -COO-L 2 -*,

[0034] L 1 represents a straight chain or branched (C1-C30) alkyl group,

[0035] L 2 represents a single bond or a straight chain or branched (C1-C30) alkyl group,

[0036] R 1 and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, R 2 represents a straight chain or branched (C1-C10) alkyl group,

[0037] B stands for Wherein, * indicates the binding site.

[0038] In one embodiment, the anti-inflammatory agent may have a structure represented by Formula 2 below.

[0039] [Chemical formula 2]

[0040]

[0041] Effects of the Invention

[0042] According to the present invention, DO3A-based substances containing gadolinium complexes, which are well-known as anti-inflammatory agents in the prior art, have a new effect of inhibiting thrombus or dissolving blood. In particular, DO3A-based substances containing gadolinium complexes can effectively inhibit platelet aggregation induced by thrombin. Therefore, the thrombus inhibitor or hemolytic agent of the present invention has the advantage that it does not need to be administered together with other thrombus inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph evaluating the platelet aggregation inhibitory effect of MBP-11902 of the present invention according to Experimental Example 1 of the present invention. In the graph, MBP-11902 is indicated as MBP-02.

[0044] Figure 2 This is a graph comparing and evaluating the platelet aggregation inhibitory effects of MBP-11902 of the present invention and NSAID and MBP-11902L as comparative examples according to Experimental Example 2 of the present invention.

[0045] In the figure, MBP-11902 and MBP-119002L are respectively indicated as MBP-02 and MBP-02L.

[0046] Figure 3 This is a diagram for evaluating the platelet activation effect of MBP-11902 of the present invention according to Example 3 of the present invention. In the figure, MBP-11902 is indicated as MBP-02.

[0047] Figure 4 This is a diagram confirming the effect of MBP-11902 of the present invention on the secretion of ATP and TXB2 of platelets according to Example 4 of the present invention. In the figure, MBP-11902 is indicated as MBP-02.

[0048] Figure 5 The effect of MBP-11902 on platelet Ca 2+ Figure 2 shows the effect of mobilization. In the figure, MBP-11902 is indicated as MBP-02. DETAILED DESCRIPTION

[0049] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. The present invention can be modified in many ways and can have many forms, so specific embodiments are illustrated in the drawings and described in detail in this specification. However, it should be understood that this does not limit the present invention to a specific disclosure method, but includes all modifications, equivalent technical solutions and alternative solutions included in the concept and technical scope of the present invention. When describing the drawings, similar components are marked with similar reference numerals.

[0050] The terms used in this application are only used to illustrate specific embodiments and are not intended to limit the present invention. Unless the context clearly indicates a different meaning, a singular expression includes a plural expression. In this application, terms such as "including" or "having" are used to specify the existence of features, steps, actions, constituent elements, parts, or combinations thereof recorded in the specification, and shall not be understood as excluding in advance the existence or additional possibility of one or more other features or steps, actions, constituent elements, parts, or combinations thereof.

[0051] Unless otherwise defined, all terms used in this application, including technical and scientific terms, have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Terms defined in commonly used dictionaries should be interpreted as having the same meaning as that in the context of the relevant art, and, unless explicitly defined in this application, should not be interpreted in an idealized or overly formalized sense.

[0052] The following is a more detailed description of the DO3A-based thrombus inhibitor or hemolytic agent containing a gadolinium complex of the present invention through specific examples and comparative examples. However, the embodiments of the present invention are only some embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0053] Example 1 <mbp-11902>

[0054] The compound used in Example 1 of the present invention (hereinafter referred to as MBP-11902) was provided by Etnova Therapeutics, Inc. MBP-11902 in Example 1 of the present invention is indicated as MBP-02 in the accompanying drawings.

[0055] Comparative Example 1 <nsaid>

[0056] The compound used in Comparative Example 1 of the present invention (hereinafter referred to as NSAID) is Diflunisal, which is provided by Etnova Therapeutics.

[0057] Comparative Example 2 <mbp-11902l>

[0058] The compound used in Comparative Example 2 of the present invention (hereinafter referred to as MBP-11902L) is a ligand compound except for gadolinium in the compound of Example 1 of the present invention, and is provided by Etnova Therapeutics. MBP-11902L in Comparative Example 2 of the present invention is indicated as MBP-02L in the accompanying drawings.

[0059] Experimental Example 1

[0060] ① Evaluation of the platelet aggregation inhibitory efficacy of MBP-11902

[0061] In order to evaluate the platelet aggregation activity of MBP-11902 according to Example 1 of the present invention, platelet aggregation was activated in rat plasma with platelet aggregation factors and so-called agonists such as thrombin, collagen, ADP (adenosine diphosphate), U46619 (thromboxane A2 agonist), and PMA (phorbol-12-myristate-13-acetate), and platelet aggregation was measured using a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA) after pretreatment with a vehicle (distilled water) and different doses (100, 300 and 500 μM) of MBP-11902. The results are shown in FIG. Figure 1 middle.

[0062] Figure 1 This is a graph evaluating the platelet aggregation inhibitory effect of MBP-11902 of the present invention according to Experimental Example 1 of the present invention.

[0063] See also Figure 1 , compared with the control group (vehicle), it was observed that MBP-11902 of the present invention effectively inhibited platelet aggregation induced by thrombin (0.05U / mL). In addition, when the platelet aggregation effect according to the concentration (25, 50, 100, 300 and 500μM) of MBP-11902 was observed, it was observed that the inhibitory effect did not differ greatly depending on the concentration. In addition, it was not observed that MBP-11902 of the present invention effectively inhibited platelet aggregation induced by collagen (1μg / mL), ADP (10μM), U46619 (1μM) and PMA (1μM).

[0064] Based on the above results, it can be seen that although MBP-11902 of the present invention was confirmed to inhibit platelet aggregation induced only by thrombin, it did not inhibit platelet aggregation in a concentration-dependent manner. This shows that although MBP-11902 showed efficacy in inhibiting platelet aggregation through a secondary signaling pathway in platelet aggregation mediated by thrombin, it has limitations in platelet aggregation through morphological deformation of platelets and primary signaling pathways.

[0065] ②Comparative evaluation of the inhibitory effects of MBP-11902, NSAIDs, and MBP-11902L on platelet aggregation

[0066] In order to re-evaluate the efficacy of MBP-11902 in Experimental Example 1 in inhibiting platelet aggregation using only thrombin as a medium, the platelet aggregation of the prototype NSAID, the intermediate MBP-11902L, and the main MBP-11902 (each 100 μM) was evaluated. For evaluation, thrombin and collagen were used to measure the values ​​according to the platelet aggregation degree using a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA), and the results are shown in Figure 2 .

[0067] Figure 2 This is a diagram for comparative evaluation of the platelet aggregation inhibitory effects of MBP-11902 of the present invention and NSAID and MBP-11902L as comparative examples according to Experimental Example 2 of the present invention.

[0068] See also Figure 2 , compared with the control group (vehicle), it was observed that NSAID (100 μM) significantly inhibited platelet aggregation induced by thrombin (0.05 U / ml) and collagen (1 μg / mL). On the other hand, compared with the control group (vehicle), it was observed that the intermediate MBP-11902L (100 μM) was ineffective in inhibiting platelet aggregation in both thrombin (0.05 U / ml) and collagen (1 μg / mL). In addition, it was confirmed that MBP-11902 of the present invention inhibited platelet aggregation induced only by thrombin.

[0069] Based on the above results, it can be seen that the prototype NSAID can significantly inhibit platelet activity, the intermediate MBP-11902L has no effect of inhibiting platelet aggregation, and the MBP-11902 of the present invention only inhibits platelet aggregation induced only by thrombin in the secondary signaling pathway. Therefore, although the prototype NSAID has an excellent effect in inhibiting platelet aggregation, it is expected to have side effects on bleeding. In addition, the MBP-11902 of the present invention shows platelet aggregation inhibition effect only through the secondary signaling pathway using only thrombin as a medium during platelet aggregation. Therefore, compared with the prototype NSAID and the intermediate MBP-11902L, platelet aggregation can be inhibited in platelet aggregation using thrombin as a medium without causing side effects on bleeding.

[0070] ③ Evaluation of platelet activation efficacy of MBP-11902

[0071] In the platelet aggregation inhibition experiment of the above-mentioned Experimental Example 1, it was confirmed that MBP-11902 has the effect of selectively inhibiting platelet aggregation on the signaling pathway using thrombin as a medium. Therefore, in this platelet activation experiment, only thrombin was used to perform the activation efficacy inhibition experiment. The function of MBP-11902 on the activation regulatory mechanism of α-granules, P-selectin, and integrin αIIBβ3, which are used as platelet activation biomarkers, in the main process of inducing a positive feedback cycle in platelet activation was evaluated by flow cytometry analysis (FCM), and the results are shown in Figure 3 .

[0072] Figure 3 This is a graph evaluating the platelet activation efficacy of MBP-11902 of the present invention according to Example 3 of the present invention.

[0073] See also Figure 3 Compared with the control group (vehicle), it was observed that MBP-11902 of the present invention significantly inhibited the activation of rat platelet integrin αIIBβ3 in a concentration (100, 300 and 500 μM) dependent manner, but no activity inhibitory effect was observed in the activities of α-granules (α-granule) and P-selectin (P-selectin).

[0074] This result indicates that MBP-11902 of the present invention selectively inhibits platelet-platelet aggregation activity by activating integrin αIIbβ3 present in the platelet cell membrane without affecting platelet granule secretion.

[0075] ④ Evaluate the inhibition of platelet activity by MBP-11902 by measuring ATP and TXB2 secretion. It is known that in the process of platelet aggregation, ATP (Adenosine triphosphate) and TXB2 (Thromboxane B2) are released, and activate the intrinsic receptors of the platelet cell wall through autocrine and paracrine, promoting platelet aggregation. Therefore, in the platelet aggregation reaction, ATP and TXB2 secretion and activation are the main mechanisms of thrombus aggregation reaction.

[0076] In the present invention, in order to confirm the ATP and TXB2 secretion activity of MBP-11902, platelet aggregation activity was increased by thrombin, and then MBP-11902 was treated with different concentrations (100, 300 and 500 μM), and the ATP and TXB2 generation concentrations were measured using luciferin / luciferase reagent (Chronolog) and TXB2 generation assay kit, and the results are shown in Figure 4 .

[0077] Figure 4 This is a diagram confirming the effect of MBP-11902 of the present invention on the secretion of ATP and TXB2 of platelets according to Example 4 of the present invention.

[0078] See also Figure 4 Compared with the control group (vehicle), it was confirmed that only in the platelets treated with MBP-11902, the ATP secretion of rat platelets was significantly inhibited in a concentration-dependent manner. On the other hand, there was no significant difference in TXB2 production. This result shows that MBP-11902 of the present invention affects platelet activity by regulating ATP secretion in platelet activity, but does not affect TXB2 production.

[0079] ⑤Evaluation of the effect of MBP-11902 on platelet Ca 2+ The impact of mobilization

[0080] Platelet agonists stimulate other receptors on platelets to secrete intracellular Ca2+ stores 2+ or accept Ca from outside the platelet 2+ , thus, leading to the Ca 2+ Mobilization (Ca 2+ mobilization). 2+ The basic mechanism of mobilization is very important for platelet activation, including platelet α-granules, P-selectin, and integrin αIIBβ3 activation regulatory mechanism. Therefore, whether MBP-11902 of the present invention regulates Ca during platelet activation was investigated. 2+ Mobilization plays an important role.

[0081] The control group (vehicle) or different doses (100, 300 and 500 μM) of MBP-11902 were induced to platelets (1×10 8 The platelets were treated with 5% paraformaldehyde (5% paraformaldehyde) and 4% paraformaldehyde (5% paraformaldehyde) for 10 minutes. The platelets were incubated with the FLIPR Calcium 5 Assay kit at 37°C for 30 minutes in a dark place, and then activated with thrombin. Cytoplasmic Ca was measured using a spectrofluorometer (spectramax I3, Molecular Devices) with an input wavelength of 485 nm and an output wavelength of 525 nm. 2+ concentration, and the results are shown in Figure 5 .

[0082] Figure 5 The effect of MBP-11902 on platelet Ca 2+ Figure 2 shows the impact of mobilization.

[0083] See also Figure 5 , it was observed that platelet Ca 2+ The mobilization of Ca was not controlled by MBP-11902. 2+ Platelet activation in response to concentration changes was not modulated by MBP-11902.

[0084] The results of Experimental Examples 1 to 5 of the present invention are summarized as follows.

[0085] The MBP-11902 of the present invention does not affect thrombin-mediated platelet activation, but significantly inhibits platelet-platelet aggregation, and therefore plays a more important role in the second platelet-platelet aggregation inhibition than the first inhibition of platelet-mediated thrombus. The MBP-11902 of the present invention does not affect the hemostatic effect, and while replacing the side effects of the existing therapeutic agent NSAID, it has an effect on thrombolysis. Therefore, the MBP-11902 of the present invention can be used as a raw material or therapeutic agent for improving blood circulation through the thrombolysis effect.

[0086] <Materials and Analysis Methods Used in Experimental Examples 1 to 5>

[0087] ①Materials

[0088] Human thrombin, thromboxan A2 analogue (U46619), PMA, PGE1, dimethyl sulfoxide (DMSO), ADP, fibrinogen, and all reagents were purchased from Sigma (St. Louis, MO). D-Phe-Pro-Arg-chloromethyl ketone (PPACK) was purchased from EMD Millipore (Billerica, MA). Phycoerythrin (PE)-conjugated isotype control IgGs, PE anti-mouse CD62P (P-selectin) antibody, and PE anti-mouse αIIbβ3 (JON / A) antibody were purchased from Emfret Analytics (Eibelstadt, Germany). FLIPR Calcium 5 Assay Kit was a Ca 5 assay kit. 2+ The indicator was purchased from Molecular Devices (Sunnyvale, CA, USA) and used in this experiment.

[0089] ② Platelet separation method

[0090] Platelets from male C57BL / 6 (6-8 weeks old) were collected from the abdominal vein of rats using a syringe pretreated with a citrate-dextrose solution (ACD, Sigma). To obtain platelet-rich plasma (PRP), centrifuge at 300 × g for 20 minutes at room temperature, then add 0.5 μM PGE1 to the plasma separated from the erythrocytes and centrifuge again at 700 × g for 4 minutes, wash the precipitated platelets with HEPES-Tyrode buffer (5mM HEPES / NaOH, pH 7.3, 5mM glucose, 136mM NaCl, 12mM NaHCO3, 2.7mM KCl) containing 10% ACD, and then centrifuge again at 700 × g for 5 minutes. The precipitated platelets were adjusted to 3 × 10 in HEPES-Tyrode buffer. 8 The concentration of cells / mL was used in this experiment.

[0091] ③ Platelet aggregation analysis method

[0092] Rat platelets were collected at 3×10 8 Cells were placed in HEPES-Tyrod buffer at a concentration of 10 cells / mL, and pretreated with distilled water and different doses (100, 300 and 500 μM) of MBP-11902 for 15 minutes at 37°C, and then activated with collagen, thrombin, ADP, U46619 (endoperoxide / thromboxane analogs), PMA (PKC activator), which are called agonists in the platelet aggregation system. Platelet aggregation was measured by inducing platelet aggregation at 1,000 rpm in a platelet aggregometer (Chronolog Corp, Havertown, PA) at 37°C.

[0093] ④ ATP secretion measurement

[0094] Rat platelets were pretreated with distilled water and different doses (100, 300, and 500 μM) of MBP-11902 at 37°C for 10 minutes and then activated with thrombin, one of the platelet aggregation agonists. The platelet activity reaction was terminated by centrifugation, and the supernatant was collected to measure the secretion of adenosine triphosphate (ATP). The ATP detection kit (Biomedical Research Service Center, Buffalo, NY) was used to detect the secretion of ATP. ATP secretion was measured using Explorer Multimode Microplate Reader (Promega).

[0095] ⑤Measure TXB2 generation

[0096] Rat platelets were pretreated with distilled water and different doses (100, 300 and 500 μM) of MBP-11902 at 37°C for 10 minutes, and then activated with thrombin, one of the platelet aggregation agonists, at 37°C. Platelet activation was stopped after 5 minutes by adding 2 mM EGTA containing 0.1 M KCl and 5 mM indomethacin. The mixture was centrifuged at 6,000 × g for 3 minutes, the supernatant was collected, and the thromboxane B2 (TXB2) generation number was measured using a TXB2 generation kit (Enzo Life Sciences, Farmingdale, NY).

[0097] ⑥Flow cytometry analysis (FCM)

[0098] P-selectin and integrin αIIBβ3 activation, which are known as biomarkers of platelet activity, were measured using a flow cytometer (Gallios, Beckman Coulter). Rat platelets were pretreated with distilled water and different doses (100, 300 and 500 μM) of MBP-11902 at 37°C for 15 minutes. For platelet activity, rat platelets were treated with thrombin at 37°C for 5 minutes, and then the activity of P-selectin and integrin αIIBβ3 was measured.

[0099] ⑦Ca 2+ Mobilization analysis

[0100] Rat platelets (1×10 8 Cells / mL) were suspended in HEPES-Tyrod buffer without CaCl2 and pretreated with distilled water and different doses (100, 300 and 500 μM) of MBP-11902 at 37°C for 10 min. For rat platelets, Ca 2+ Staining (FLIPR Ca 2+ 5analysis kit) for staining, and then activated with 0.05U / mL thrombin. A spectrofluorimeter (Spectramax I3, Molecular Devices) was used to measure the Ca in platelets at a wavelength of 505nm. 2+ level.

[0101] ⑧Statistical processing

[0102] Data were statistically analyzed using GraphPad Prism 5. Statistical significance was analyzed by multiple group ANOVA and Dunnett's test for comparisons of multiple groups and Student's t-test for comparisons of two groups. P values ​​less than 0.05 were considered significant.

[0103] Although the present invention has been described above with reference to the preferred embodiments, it will be appreciated by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as described in the claims. < / nsaid>

Claims

1. A thrombus inhibitor or hemolytic agent having a structure represented by the following formula 1: [Chemical formula 1] In Chemical Formula 1, A represents *-(CH2) n -A 1 -*, n represents any integer from 0 to 5, A 1 Indicates *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-* or *-O-*, Linker indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2 -OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -* or *-L 1 -COO-L 2 -*, L 1 represents a straight chain or branched (C1-C30) alkyl group, L 2 represents a single bond or a straight chain or branched (C1-C30) alkyl group, R 1 and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, R 2 represents a straight chain or branched (C1-C10) alkyl group, B stands for in, *Indicates binding site.

2. The thrombus inhibitor or hemolytic agent according to claim 1, characterized in that: The thrombus inhibitor or hemolytic agent has a structure represented by the following Formula 2: [Chemical formula 2] 3. The thrombus inhibitor or hemolytic agent according to claim 1, characterized in that: The thrombus inhibitor or hemolytic agent inhibits platelet aggregation induced by thrombin.

4. The thrombus inhibitor or hemolytic agent according to claim 3, characterized in that: The thrombus inhibitor or hemolytic agent inhibits integrin αIIBβ3 activation in a concentration-dependent manner.

5. The thrombus inhibitor or hemolytic agent according to claim 3, characterized in that: The thrombus inhibitor or hemolytic agent inhibits the secretion of ATP (Adenosine triphosphate).

6. An anti-inflammatory agent, characterized in that It has a structure represented by the following formula 1 and has the property of inhibiting thrombus or dissolving blood: [Chemical formula 1] In Chemical Formula 1, A represents *-(CH2) n -A 1 -*, n represents any integer from 0 to 5, A 1 Indicates *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-* or *-O-*, Linker indicates *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2 -OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -* or *-L 1 -COO-L 2 -*, L 1 represents a straight chain or branched (C1-C30) alkyl group, L 2 represents a single bond or a straight chain or branched (C1-C30) alkyl group, R 1 and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, R 2 represents a straight chain or branched (C1-C10) alkyl group, B stands for Wherein, * indicates the binding site.

7. The anti-inflammatory agent according to claim 6, characterized in that The anti-inflammatory agent has a structure represented by the following Formula 2: [Chemical formula 2]

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