Thrombotic inhibitors or hemolytic agents based on DO3A containing gadolinium complexes

Abstract

The present invention has discovered that DO3A-based substances containing gadolinium complexes, which are conventionally used as anti-inflammatory agents, have a novel effect of inhibiting thrombin-induced thrombosis or dissolving blood, without being involved in the platelet action function of stopping bleeding at an injury or wound site, and discloses the applicability of DO3A-based substances containing gadolinium complexes to novel uses as thrombosis inhibitors or blood coagulants.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition based on DO3A containing a gadolinium complex, and more specifically, to an invention that provides a novel effect of inhibiting thrombosis or dissolving blood in a pharmaceutical composition conventionally known as an anti-inflammatory agent. [Background technology]

[0002] Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the COX-1 and COX-2 enzymes, which are involved in prostaglandin synthesis. This action produces analgesic, anti-inflammatory, and antipyretic effects. COX-1 activates prostaglandins and thromboxane A2, playing a protective role in protecting the gastric mucosa, maintaining kidney function, and stabilizing platelets. COX-2 activates inflammation mediators and cytokines in various tissues, including vascular intima cells, thereby generating pain and inflammation.

[0003] NSAIDs are classified into non-selective NSAIDs and COX-2 selective NSAIDs based on their selectivity for COX-1. Non-selective NSAIDs inhibit both COX-1 and COX-2, which has the fatal weakness of frequently causing gastrointestinal side effects such as peptic ulcers due to impaired gastric mucosal protection and antiplatelet effects. Subsequently, COX-2 selective NSAIDs emerged with the expectation that they could resolve the various side effects caused by blocking COX-1 while maintaining anti-inflammatory and analgesic effects by selectively inhibiting only COX-2.

[0004] While COX-2 selective NSAIDs are known to reduce the incidence of gastrointestinal complications, there are still logical issues regarding cardiovascular and new toxic side effects. When an NSAID inhibits COX-2, the production of prostacyclin is suppressed, while the antiplatelet effect decreases, sodium, and water retention occur, and the platelet coagulation effect by COX-1 is further promoted, increasing the risk of thrombosis and embolism, which increases the occurrence of cardiovascular events.

[0005] On the other hand, 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 described that a compound in which diflunisal is bound to DO3A containing a Gd complex and a compound in which diflunisal is bound to DO3A can be used as a selective COX-2 inhibitor and an anti-inflammatory agent, respectively. Based on the contents of these two patents, it can be seen that a compound in which diflunisal is bound to DO3A is well-known to act as a selective COX-2 inhibitor and an anti-inflammatory agent, among which it acts as an anti-inflammatory agent for cerebral inflammatory diseases. However, as can be seen from the content of Korean Patent No. 10-2020-0119574, the effect as an anti-inflammatory agent has been proven, but the effect on thrombus inhibition has not been disclosed.

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0007] The thrombus inhibitor or thrombolytic agent for one object of the present invention contains a compound having a structure represented by the following Chemical Formula 1. The compound having the structure of the following Chemical Formula 1 is a substance that has been conventionally known as an anti-inflammatory agent. In the prior art, cardiovascular side effects were resolved by co-administering the compound with other thrombus inhibitors. However, the inventor of the present invention has found that the compound having the structure of the following Chemical Formula 1 provides a new effect in suppressing thrombus or lysing blood in addition to the anti-inflammatory agent effect, thereby providing that the compound having the structure of the following Chemical Formula 1 can be used for a new pharmaceutical use as a thrombus inhibitor or a thrombolytic agent.

[0008]

Chemical Formula

[0009] In the above Chemical Formula 1, A represents *-(CH2) n -A 1 -*, n represents an integer of any one of 0 to 5, A 1 represents *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-*, or *-O-*, Linker represents *-L 1 -NHCO-L 2 -*, *-L 1 -O-R 2 -O-L 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 [[ID=…]]-*, or *-L 1 -COO-L 2 -*, L 1 represents a linear or branched (C1-C30) alkyl, L 2 represents a single bond, or a linear or branched (C1-C30) alkyl, R 1and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, and R 2 This represents a linear or branched (C1-C10) alkyl group. B is

[0010] [ka]

[0011] This indicates the linkage site, where * represents the binding site.

[0012] Preferably, the thrombosis inhibitor or blood solvent of the present invention has a structure represented by the following chemical formula 2, in which diflunisal is bonded to DO3A containing a gadolinium complex.

[0013] [ka]

[0014] When blood vessels are damaged in the body and bleeding occurs, hemostasis is achieved through primary and secondary hemostasis processes. Primary hemostasis is platelet-mediated hemostasis, where platelets adhere to and aggregate at the site of the damaged blood vessel, while secondary hemostasis is hemostasis by coagulation factors, where activation of coagulation factors forms hard fibrin on the aggregated platelets, completely stopping the bleeding. In primary hemostasis, platelets change morphologically from a disc shape to a spherical shape as they adhere to and aggregate with the collagen of the blood vessels. The adhered platelets are activated and contain calcium (Ca 2+The body releases granular substances such as ) and ADP (adenosine diphosphate). The ADP secreted here binds to surrounding platelets and activates them, causing platelet aggregation. Secondary hemostasis is the formation of hard fibrin through the activation of blood coagulation factors. Activation of blood coagulation factors generates thrombin via intrinsic and extrinsic pathways, and thrombin acts on fibrinogen to form fibrin polymer. Fibrin or fibrin polymer binds to the primary hemostatic plug, which consists of platelet adhesion and aggregation in primary hemostasis, to form an even firmer and harder secondary hemostatic plug, thus achieving complete hemostasis.

[0015] The thrombosis inhibitor or hemolytic agent of the present invention is characterized by its ability to suppress platelet aggregation induced by thrombin, which is a secondary hemostatic agent. Rather than suppressing platelet adhesion at the injury site, which occurs in primary hemostasis, the thrombosis inhibitor or hemolytic agent of the present invention has the effect of suppressing platelet aggregation induced by thrombin, which occurs in secondary hemostasis.

[0016] In one embodiment, the thrombotic agent or hemolytic agent can control integrin αIIBβ3 activation. Integrin αIIBβ3 is a protein involved in adhesion and mediates the interaction between platelet aggregation and other adjacent tissues. The thrombotic agent or hemolytic agent of the present invention can suppress integrin αIIBβ3 activation in a concentration-dependent manner. In the present invention, "concentration-dependent" means that the degree of effect increases as the concentration increases, or decreases as the concentration decreases. That is, the higher the concentration of the thrombotic agent or hemolytic agent of the present invention, the more effectively it can suppress integrin αIIBβ3 activity involved in platelet aggregation.

[0017] In one embodiment, the thrombotic inhibitor or hemolytic agent can suppress the secretion of adenosine triphosphate (ATP). Adenosine triphosphate is a substance released by platelets while they are activated and can bind to P2Y2 receptors on the surface of vascular endothelial cells. The present invention can influence platelet activity by suppressing ATP secretion.

[0018] An anti-inflammatory agent for another purpose of the present invention is characterized by having a structure represented by the following chemical formula 1 and possessing thrombosis-inhibiting or hemolytic properties. Since the anti-inflammatory agent has thrombosis-inhibiting or hemolytic properties, it has the advantage of not requiring concomitant administration with other drugs for thrombosis inhibition.

[0019] [ka]

[0020] In the aforementioned chemical formula 1, A is *-(CH2) n -A 1 -* indicates n represents an integer between 0 and 5. A 1 *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-*, or *-O-* Linker is *-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 -* indicates L 1 This represents a linear or branched (C1-C30) alkyl group. L2 This represents a single bond, or a linear or branched (C1-C30) alkyl group. R 1 and R 3 Each independently represents hydrogen or a linear or branched (C1-C10) alkyl group, and R 2 This represents a linear or branched (C1-C10) alkyl group. B is

[0021] [ka]

[0022] This indicates the linkage site, where * represents the binding site.

[0023] In one embodiment, the anti-inflammatory agent may have a structure represented by the following chemical formula 2.

[0024] [ka] [Effects of the Invention]

[0025] According to the present invention, a DO3A-based substance containing a gadolinium complex, which has been known as an anti-inflammatory agent in the prior art, has a novel effect of inhibiting thrombosis or dissolving blood. In particular, a DO3A-based substance containing a gadolinium complex can effectively suppress thrombin-induced platelet aggregation. Therefore, the thrombosis inhibitor or blood-dissolving agent of the present invention has the advantage of not requiring concomitant administration with other thrombosis inhibitors. [Brief explanation of the drawing]

[0026] [Figure 1] This figure shows the evaluation of the platelet aggregation inhibitory efficacy of MBP-11902 according to Experimental Example 1 of the present invention. In the figure, MBP-11902 is indicated as MBP-02. [Figure 2]This figure shows the comparative evaluation of the platelet aggregation inhibitory efficacy of MBP-11902 of the present invention and comparative examples NSAID and MBP-11902L, according to Experimental Example 2 of the present invention. In the figure, MBP-11902 and MBP-11902L are indicated as MBP-02 and MBP-02L, respectively. [Figure 3] This figure shows the evaluation of the platelet-activating efficacy of MBP-11902 according to Example 3 of the present invention. In the figure, MBP-11902 is indicated as MBP-02A. [Figure 4] This figure shows the effects of MBP-11902 of the present invention on platelet ATP and TXB2 secretion according to Example 4 of the present invention. In the figure, MBP-11902 is indicated as MBP-02. [Figure 5] This figure shows the effect of MBP-11902 of the present invention on platelet Ca2+ behavior (mobilization) according to Experimental Example 3 of the present invention. In the figure, MBP-11902 is indicated as MBP-02. [Modes for carrying out the invention]

[0027] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The present invention can be modified in various ways and may take many forms; specific embodiments are illustrated in the drawings and described in detail in the text. However, this should be understood not as an attempt to limit the invention to any particular disclosure, but rather as encompassing all modifications, equivalents, or substitutions that fall within the spirit and technical scope of the invention. Similar reference numerals have been used for similar components in the description of each drawing.

[0028] The terms used in this application are used solely to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, terms such as “includes” or “having” are intended to specify the existence of features, stages, operations, components, parts, or combinations thereof described in the specification, and should be understood not to preemptively exclude the existence or possibility of adding one or more other features, stages, operations, components, parts, or combinations thereof.

[0029] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as they would be generally understood by a person of ordinary skill in the art to which this invention pertains. Terms as defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an ideal or overly formal sense unless explicitly defined herein.

[0030] The following describes in more detail the DO3A-based antithrombotic or hemolytic agent containing the gadolinium complex of the present invention through specific examples and comparative examples. However, the examples of the present invention represent only a partial embodiment of the present invention, and the scope of the present invention is not limited to the examples described below.

[0031] Example 1 <mbp-11902> The compound used in Example 1 of the present invention (hereinafter referred to as MBP-11902) was supplied by Etnova Theraputics. In Example 1 of the present invention, MBP-11902 is denoted entirely as MBP-02 in the drawings.

[0032] Comparative Example 1 <nsaid> The compound used in Comparative Example 1 of the present invention (hereinafter referred to as NSAID) was diflunisal, supplied by Etnova Therapeutics.

[0033] Comparative Example 2 <mbp-11902l> The compound used in Comparative Example 2 of the present invention (hereinafter referred to as MBP-11902L) is a ligand compound from which gadolinium has been removed from the compound in Example 1 of the present invention, and was provided by Etnova Theraputics. MBP-11902L in Comparative Example 2 of the present invention is denoted entirely as MBP-02L in the drawings.

[0034] Experimental Example 1 (1) Evaluation of the efficacy of MBP-11902 in inhibiting platelet aggregation. To evaluate the platelet aggregation activity of MBP-11902 according to Example 1 of the present invention, rat plasma was pre-treated with a vehicle (distilled water) and various volumes (100, 300, and 500 μM) of MBP-11902 to activate platelet aggregation. The results are shown in Figure 1.

[0035] Figure 1 is a diagram for evaluating the platelet aggregation inhibitory efficacy of MBP-11902 according to Experimental Example 1 of the present invention.

[0036] Referring to Figure 1, it was observed that MBP-11902 of the present invention effectively inhibited thrombin (0.05 U / mL)-induced platelet aggregation compared to the control group (vehicle). On the other hand, when observing the platelet aggregation effect at different concentrations of MBP-11902 (25, 50, 100, 300, and 500 μM), no significant difference in inhibitory effect with respect to concentration was observed. Furthermore, MBP-11902 of the present invention showed no effect in inhibiting platelet aggregation induced by collagen (1 μg / mL), ADP (10 μM), U46619 (1 μM), and PMA (1 μM).

[0037] In summary, the results confirm that MBP-11902 of the present invention inhibits platelet aggregation induced solely by thrombin, but it is not able to suppress platelet aggregation in a concentration-dependent manner. This suggests that while MBP-11902 exhibits efficacy in suppressing platelet aggregation via a secondary signaling pathway in thrombin-mediated platelet aggregation, there are limitations in its ability to induce platelet aggregation through morphological deformation of platelets and the primary signaling pathway.

[0038] (2) Comparative evaluation of the platelet aggregation inhibitory efficacy of MBP-11902, NSAIDs, and MBP-11902L In Experimental Example 1, to re-evaluate the platelet aggregation inhibitory efficacy of MBP-11902 using thrombin alone, the platelet aggregation activity was evaluated against the proto-NSAID, the intermediate MBP-11902L, and the active ingredient MBP-11902 (100 μM each). The evaluation was performed using thrombin and collagen, measuring the degree of platelet aggregation with a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA), and the results are shown in Figure 2.

[0039] Figure 2 is a diagram for comparing and evaluating the platelet aggregation inhibitory efficacy of MBP-11902 of the present invention with comparative examples NSAID and MBP-1192L, according to Experimental Example 2 of the present invention.

[0040] Referring to Figure 2, NSAID (100 μM) was observed to significantly inhibit platelet aggregation induced by thrombin (0.05 U / ml) and collagen (1 μg / mL) compared to the control group (vehicle). On the other hand, the intermediate MBP-11902L (100 μM) was observed to have no effect on inhibiting platelet aggregation in both thrombin (0.05 U / ml) and collagen (1 μg / mL) compared to the control group (vehicle). Meanwhile, MBP-11902 of the present invention was confirmed to inhibit platelet aggregation induced solely by thrombin.

[0041] In summary, the results show that the original NSAID significantly suppresses platelet activity, the intermediate MBP-11902L has no efficacy in suppressing platelet aggregation, and the MBP-11902 of the present invention inhibits platelet aggregation induced solely by thrombin only through the secondary signaling pathway. Therefore, while the original NSAID is excellent at suppressing platelet aggregation, it is thought to be associated with bleeding side effects. On the other hand, the MBP-11902 of the present invention exhibits platelet aggregation inhibitory efficacy only through the secondary signaling pathway mediated solely by thrombin during platelet aggregation, and is thought to suppress platelet aggregation without bleeding side effects in thrombin-mediated platelet aggregation compared to the original NSAID and the intermediate MBP-11902L.

[0042] (3) Evaluation of the platelet activation efficacy of MBP-11902 In the platelet aggregation inhibition experiment described in Experimental Example 1, MBP-11902 selectively inhibited platelet aggregation in the thrombin-mediated signaling pathway. Therefore, in this platelet activation experiment, we used only thrombin to perform an activation efficacy inhibition experiment. The efficacy of MBP-11902 on the regulatory mechanisms of α-granule, P-selectin, and integrin αIIBβ3 activation, which are used as platelet activity biomarkers in the main process of inducing a positive feedback cycle through platelet activation, was evaluated using flow cytometry analysis (FCM), and the results are shown in Figure 3.

[0043] Figure 3 is a diagram for evaluating the platelet-activating efficacy of MBP-11902 of the present invention according to Example 3 of the present invention.

[0044] Referring to Figure 3, it was observed that MBP-11902 of the present invention significantly suppressed rat platelet integrin αIIBβ3 activation in a concentration (100, 300, and 500 μM) dependent manner compared to the control group (vehicle), but it was observed that there was no inhibitory effect on the activity of α-granules and P-selectin.

[0045] These results suggest that MBP-11902 of the present invention does not affect platelet granule secretion, but selectively suppresses platelet-platelet aggregation activity by activating integrin αIIBβ3 present in the platelet cell membrane.

[0046] (4) Evaluation of platelet activity inhibition by MBP-11902 by measuring ATP and TXB2 secretion. During platelet aggregation, ATP (Adenosine triphosphate) and TXB2 (Thromboxane B2) are released, activating intrinsic receptors on the outer wall of platelet cells via autocrine and paracrine mechanisms, thereby promoting platelet aggregation. Therefore, ATP and TXB2 secretion and activation are known to be the main mechanisms of thrombosis in the platelet aggregation reaction.

[0047] In this invention, to confirm the ATP and TXB2 secretion activity of MBP-11902, platelet aggregation activity was increased with thrombin, and then MBP-11902 was treated at different concentrations (100, 300, and 500 μM). The ATP and TXB2 production concentrations were measured using a luciferin / luciferase reagent (Chronolog) and a TXB2 production analysis kit, and the results are shown in Figure 4.

[0048] Figure 4 is a diagram illustrating the effects of MBP-11902 of the present invention on platelet ATP and TXB2 secretion according to Example 4 of the present invention.

[0049] Referring to Figure 4, it was confirmed that MBP-11902 significantly suppressed rat platelet ATP secretion in a concentration-dependent manner only in platelets treated with MBP-11902 compared to the control group (vehicle). On the other hand, no significant difference was observed in TXB2 production. These results indicate that MBP-11902 of the present invention affects platelet activity through ATP secretion regulation, but does not affect TXB2 production.

[0050] (5) Platelet Ca of MBP-11902 2+ Evaluation of the impact on behavior (mobilization) Platelet agonists stimulate other receptors on platelets to release the Ca2+ stored in cells. 2+ To secrete Ca from outside the platelet, 2+ By accepting Ca in the platelet cytoplasm, 2+ Behavior (Ca 2+ (mobilization) occurs. 2+ The basic mechanism of its behavior is important for platelet activation, including platelet α-granule, P-selectin, and integrin αIIBβ3 activation regulatory mechanisms. Therefore, MBP-11902 of the present invention is important for Ca activation during platelet activation. 2+ We investigated whether it plays an important role in the behavior.

[0051] Rat platelets (1 x 10) 8 Platelets were treated with MBP-11902 in a control group (vehicle) or in various volumes (100, 300, and 500 μM) at 37°C for 10 minutes. After incubation with the FLIPR Calcium 5 Assay kit at 37°C for 30 minutes in a light-blocked environment, the platelets were activated using thrombin. 2+ The concentration was measured using a fluorescence spectrophotometer (Spectramax I3, Molecular Devices) with input and output wavelengths of 485 nm and an emission wavelength of 525 nm, and the results are shown in Figure 5.

[0052] Figure 5 shows platelet Ca of MBP-11902 according to the present invention, according to experimental example 3 of the present invention. 2+ This diagram is used to evaluate the impact on behavior (mobilization).

[0053] Referring to Figure 5, the Ca of platelets 2+ The behavior was observed to be uncontrolled by MBP-11902. Such results suggest that thrombin-induced Ca 2+ This suggests that platelet activation induced by concentration changes is not regulated by MBP-11902.

[0054] The results of experimental examples 1 to 5 of the present invention can be summarized as follows:

[0055] The MBP-11902 of the present invention does not affect thrombin-mediated platelet activity, but significantly inhibits platelet-platelet aggregation, thus playing a more important role in secondary platelet-platelet aggregation inhibition than in primary platelet-mediated thrombosis inhibition. Furthermore, the MBP-11902 of the present invention does not affect hemostatic activity and has been confirmed to have thrombolytic efficacy while replacing the side effects of existing NSAIDs. Therefore, the MBP-11902 of the present invention can be applied as a blood circulation improving agent or therapeutic agent due to its thrombolytic efficacy.

[0056] <Materials and analytical methods for experimental examples 1-5> (1)Materials 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). Ca 2+ The FLIPR Calcium Assay Kit, used as the benchmark, was purchased from Molecular Devices (Sunnyvale, CA, USA) and used in this experiment.

[0057] (2) Platelet isolation method Platelets from male C57BL / 6 rats (6-8 weeks old) were collected from the abdominal vein using a syringe pre-treated with citrate-dextrose solution (ACD, sigma). To obtain platelet-rich plasma (PRP), the plasma was centrifuged at 300×g for 20 minutes at room temperature. After separating the plasma from the red blood cells, 0.5 μM PGE1 was added and the plasma was recentrifuged at 700×g for 4 minutes. The precipitated platelets were washed with HEPES-Tyrode buffer containing 10% ACD (5 mM HEPES / NaOH, pH 7.3, 5 mM glucose, 136 mM NaCl, 12 mM NaHCO3, 2.7 mM KCl), and then centrifuged again at 700×g for 5 minutes. The precipitated platelets were hydrated in HEPES-Tyrode buffer for 3 × 10⁶ minutes. 8 After adjusting the concentration to cells / mL, it was used in this experiment.

[0058] (3) Platelet aggregation analysis method Rad platelets 3 x 10 8 The cells were placed in HEPES-Tyrode's buffer at a concentration of cells / mL and pretreated with a vehicle (distilled water) and various volumes (100, 300, and 500 μM) of MBP-11902 at 37°C for 15 minutes. The cells were then activated with collagen, thrombin, ADP, U46619 (endoperoxide / thromboxane analogs), and PMA (PKC activator), which are known as agonists (promoters) in platelet aggregation systems. Platelet aggregation was induced and measured using a platelet aggregation system (Chronolog Corp, Havertown, PA) at 37°C at 1,000 rpm.

[0059] (4)ATP secretion measurement Rad platelets were pretreated at 37°C for 10 minutes with a vehicle (distilled water) and various volumes (100, 300, and 500 μM) of MBP-11902, and then activated at 37°C with thrombin, a platelet aggregation agonist. The platelet activation reaction was terminated by centrifugation, and the supernatant was collected to measure adenosine triphosphate (ATP) secretion. ATP secretion was measured using an ATP analysis kit (Biomedical Research Service Center, Buffalo, NY) with a GloMax® Explorer Multimode Microplate Reader (Promega).

[0060] (5) Measurement of TXB2 generation Rad platelets were pretreated at 37°C for 10 minutes with a vehicle (distilled water) and various volumes (100, 300, and 500 μM) of MBP-11902, and then activated with thrombin, a platelet aggregation agonist, at 37°C. Platelet activity was interrupted 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, and the supernatant was collected. Thromboxane B2 (TXB2) generation was measured using a TXB2 generation kit (Enzo Life Sciences, Farmingdale, NY).

[0061] (6) Flow cytometry analysis (FCM) P-selectin and integrin αIIBβ3 activation, known biomarkers of platelet activity, were measured using a flow cytometer (Gonalos, Beckman Coulter). Rad platelets were pretreated at 37°C for 15 minutes with a vehicle (distilled water) and various volumes (100, 300, and 500 μM) of MBP-11902. For platelet activity, Rad platelets were treated with thrombin at 37°C for 5 minutes, after which activity was measured using P-selectin and integrin αIIBβ3.

[0062] (7) Ca 2+ Behavioral analysis Rad platelets (1 x 10) 8 Platelets (cells / mL) were suspended in CaCl2-free HEPES-Tyrode's buffer and pretreated with distilled water and various volumes (100, 300, and 500 μM) of MBP-11902 at 37°C for 10 minutes. Rad platelets were left in the dark for 30 minutes. 2+ Staining (FLIPR Ca 2+ Platelet calcium was stained using the analysis kit and activated with 0.05 U / mL thrombin. 2+ The levels were measured using a 505 nm wavelength spectrofluorescence analyzer (Spectramax I3, Molecular Devices).

[0063] (8) Statistical processing Statistical analysis of the data was performed using GraphPad Prism 5. Statistical significance was analyzed using multi-group ANOVA, Dunnett's test for comparisons of multiple groups, and Student's t-test for comparisons of two groups. A p-value lower than 0.05 was considered statistically significant.

[0064] Although preferred embodiments of the present invention have been described above with reference to the present invention, those skilled in the art will understand that the present invention can be modified and altered in various ways without departing from the spirit and scope of the invention as described in the appended claims. < / nsaid>

Claims

1. A thrombosis inhibitor containing the compound represented by the following chemical formula 2 as an active ingredient. 【Chemistry 1】

2. The thrombosis inhibitor according to claim 1, characterized by inhibiting platelet aggregation induced by thrombin.

3. The thrombosis inhibitor according to claim 2, characterized in that it inhibits integrin αIIBβ3 activation in a concentration-dependent manner.

4. The thrombosis inhibitor according to claim 2, characterized by suppressing the secretion of ATP (adenosine triphosphosphate).

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