Pharmaceutical composition for preventing or treating thrombotic diseases, comprising pyrazole derivative

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating thrombotic diseases, the pharmaceutical composition comprising a compound of chemical formula 1 or an acceptable salt thereof; and a health functional food composition or cosmetic composition which is for improving blood circulation and comprises same Specifically, in the present invention, the compound of chemical formula 1 was found to simultaneously inhibit NOX1 and NOX2 and thus be highly effective in inhibiting platelet aggregation and thrombus formation.

Description

Pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a pyrazol derivative

[0001] [Cross-reference to related applications]

[0002] This application claims priority to Korean Patent Application No. 10-2024-0191803 filed on December 19, 2024 and Korean Patent Application No. 10-2025-0036760 filed on March 21, 2025, the entire contents of which are incorporated by reference into this application.

[0003] The present disclosure relates to a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a pyrazol derivative.

[0004]

[0005] As a component of the human body, blood possesses various important functions, including the transport of oxygen, nutrients, and waste products, buffering, maintaining body temperature, regulating osmotic pressure and ion balance, maintaining constant fluid levels, regulating humor, maintaining and regulating blood pressure, and providing biological defense. Normal blood circulation is facilitated by the complementary regulation of the blood coagulation and thrombolytic systems within the body. Among these, the mechanism of the blood coagulation system is reported to involve platelets adhering to and aggregating on the blood vessel walls to form a platelet thrombus, after which the blood coagulation system is activated to form a fibrin thrombus centered around the platelet aggregate.

[0006] Platelets are the smallest blood cells lacking a nucleus; they are produced by the rupture of megakaryocytes, which are precursors within the bone marrow, and are disc-shaped cells with a diameter of 2–5 μm and a thickness of approximately 0.5 μm. Their lifespan is 7–10 days, and the number of platelets in the blood of a healthy person ranges from 1.5 to 4 × 10⁶. 9The count is platelets / mL. Platelets circulate within blood vessels in an inactive state; however, when collagen and von Willebrand factor—exposed upon damage to the inner wall of blood vessels—bind through their respective receptors on the cell membrane, activation begins, and platelets play a central role in the hemostasis process through the formation of thrombi. If the platelet count is low or activity is reduced, hemostasis is not achieved smoothly; conversely, an excessive number or overactivation can induce thrombus formation even within intact, normal blood vessels, leading to thrombosis that inhibits blood circulation. Therefore, the search for substances that inhibit platelet activation and the study of their mechanisms hold significant importance for the prevention and treatment of thrombotic diseases.

[0007] Existing treatments for thrombotic diseases primarily rely on platelet activation inhibitors (e.g., aspirin, clopidogrel) and anticoagulants (e.g., warfarin, heparin). While these drugs prevent thrombus formation by inhibiting platelet aggregation and coagulation processes, they often carry side effects such as a risk of bleeding. Furthermore, some existing drugs have limitations in that they may not effectively inhibit thrombus formation caused by various factors, as their action is localized to specific enzymes or receptors.

[0008]

[0009] In one aspect, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a compound of the following formula 1 or a pharmaceutically acceptable salt thereof:

[0010] [Chemical Formula 1]

[0011] .

[0012] In another aspect, the present invention aims to provide a health functional food composition for improving blood circulation comprising a compound of Formula 1 or a food-grade acceptable salt thereof.

[0013] In another aspect, the present invention aims to provide a cosmetic composition for improving blood circulation comprising a compound of Formula 1 or a cosmetically acceptable salt thereof.

[0014]

[0015] In one aspect, the present invention may provide a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a compound of the following formula 1 or a pharmaceutically acceptable salt thereof:

[0016] [Chemical Formula 1]

[0017] .

[0018] In an exemplary embodiment, the compound may be a pyrazole derivative.

[0019] In an exemplary embodiment, the compound in the composition can inhibit the activation of platelets or the formation of blood clots.

[0020] In an exemplary embodiment, the composition may have one or more of the following features: (a) inhibition of reactive oxygen species (ROS) production in platelets by collagen stimulation, (b) inhibition of the GPVI signaling pathway, (c) inhibition of one or more tyrosine phosphorylation selected from the group consisting of Syk, LAT, Vav1, Btk, and PLCγ2, and (d) cytoplasmic calcium ions (Ca 2+ (e) reduction in concentration, (e) exposure to P-selectin or inhibition of integrin αIIbβ₃ activation, (f) inhibition of platelet adhesion to collagen.

[0021] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0022] In an exemplary embodiment, the composition may not inhibit the hemostatic action of blood.

[0023] In an exemplary embodiment, the thrombotic disease may be selected from the group consisting of arterial thrombosis, venous thrombosis, coronary artery thrombosis, deep vein thrombosis, cerebral artery thrombosis, peripheral vascular thrombosis, arterial embolism, venous embolism, renal embolism, chronic arterial occlusion, thrombotic microangiopathy, pulmonary infarction, cerebral infarction, cerebral embolism, and thrombophlebitis.

[0024] In another aspect, the present invention may provide a health functional food composition for improving blood circulation comprising a compound of Formula 1 or a food-grade acceptable salt thereof.

[0025] In an exemplary embodiment, the composition may inhibit ROS generation by platelets; activation of platelets; or formation of thrombi.

[0026] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0027] In another aspect, the present invention aims to provide a cosmetic composition for improving blood circulation comprising a compound of Formula 1 or a cosmetically acceptable salt thereof.

[0028] In an exemplary embodiment, the composition may inhibit ROS generation by platelets; activation of platelets; or formation of thrombi.

[0029] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0030]

[0031] The composition of the present invention has excellent preventive or therapeutic effects for thrombotic diseases.

[0032] The composition of the present invention has an excellent effect of inhibiting ROS production in platelets.

[0033] The composition of the present invention has excellent effects in inhibiting collagen and thrombin-induced platelet aggregation.

[0034] The composition of the present invention inhibits platelet aggregation and thrombus formation without inhibiting hemostatic action, so it can reduce the risk of bleeding that may occur when administering drugs to patients.

[0035] The composition of the present invention has an excellent blood circulation improvement effect.

[0036]

[0037] Figure 1 is an image summarizing the mechanism of antiplatelet action of APX-115 in collagen-stimulated platelets (Btk, Bruton's tyrosine kinase; cPLA2, cytosolic phospholipase A2; GPVI, glycoprotein VI; LAT, linker for the activation of T cells; NOX, NADPH oxidase; PKC, protein kinase C; PKG, protein kinase G; PLCγ2, phospholipase Cγ2; PS, phosphatidylserine; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species; sGC, soluble guanylate cyclase; Syk, spleen tyrosine kinase; TXA2, thromboxane; VASP, vasodilator-stimulated phosphoprotein).

[0038] Figures 2a to 2c show results demonstrating that APX-115 can inhibit collagen-induced platelet aggregation and ROS generation. (Figure 2a) Washed platelets from a healthy donor (5 x 10⁶ 8( / mL) was stimulated with 10 μg / mL collagen after incubating with a vehicle (0.5% DMSO) or a specified concentration of APX-115 for 10 minutes. Platelet aggregation was measured by turbidimetry and expressed as the percentage of maximum aggregation. The top panel shows representative traces of aggregation. Quantitative data are mean ± standard deviation obtained from four donors. (Figs. 2b and 2c) After CM-H2DCFDA loading (Fig. 2b) or in the presence of 1 μM DCFH2 (Fig. 2c), washed platelets were treated as in Fig. 2a. Fluorescence changes were measured by a fluorescence meter under constant stirring conditions. All top panels show representative traces. AU is an arbitrary unit. All quantitative data are mean ± standard deviation. Corresponding p-values ​​for significance are as indicated: **p < 0.01, ***p < 0.001.

[0039] Figures 3a and 3b are results showing that APX-115 can inhibit GPVI signaling induced by collagen stimulation. Washed platelets (5 x 10⁶ 8 Platelets (μg / mL) were incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, followed by stimulation with 10 μg / mL collagen for 2 minutes. Platelet lysates were used for immunoblot analysis to measure pan-protein tyrosine phosphorylation (Fig. 3a) and phosphorylation of splenic tyrosine kinase (Syk), T cell activation linkage protein (LAT), Vav1, and Bruton's tyrosine kinase (Btk) (Fig. 3b). The left panel shows representative immunoblot analyses, with the locations of molecular weight (kDa) markers indicated. Quantitative data are the mean ± standard deviation of signal intensity. Corresponding p-values ​​for significance are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

[0040] Figures 4a to 4c show results demonstrating that APX-115 can inhibit collagen-induced PLCγ2 activation and Ca2+ mobilization. (Figure 4a) Washed platelets (5 x 10⁶ 8 Platelets ( / mL) were incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, followed by stimulation with 10 μg / mL collagen for 2 minutes. Platelet lysates were used for immunoblot analysis to measure PLCγ2 phosphorylation. The left panel shows a representative immunoblot analysis, with the locations of molecular weight (kDa) markers indicated. Quantitative data are the mean ± standard deviation of signal intensity. (Figs. 4b and 4c) Fluo-3-AM loaded platelets (5 x 10⁶ 8 Fluo-3 (μg / mL) was stimulated with 10 μg / mL collagen after incubating with 0.5 mM EGTA (Fig. 4b) or 1 mM external CaCl2 (Fig. 4c) for 2 minutes, followed by incubation with a vehicle or specified concentration of APX-115 for 10 minutes. Fluo-3 fluorescence was monitored in arbitrary units (AU). The left panel shows representative traces. Quantitative data are the mean ± standard deviation of the maximum fluorescence intensity of Fluo-3. Corresponding p-values ​​for significance are indicated: *p ​​< 0.05, **p < 0.01, ***p < 0.001.

[0041] Figures 5a and 5b are results showing that APX-115 can inhibit collagen-induced platelet granule release. Washed platelets (5 x 10⁶ 8( / mL) was incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, followed by stimulation with 10 μg / mL collagen for 5 minutes. (Fig. 5a) Platelet samples were used to measure platelet dense granule release by detecting ATP luminescence using the luciferin-luciferase reagent. Quantitative data are mean ± standard deviation. (Fig. 5b) Platelets were incubated with a PE-labeled anti-P-selectin antibody (CD62P-PE), after which the expression of P-selectin on the platelet surface was monitored via flow cytometry as an indicator of α-granule release. The left panel shows representative histograms. Quantitative data are mean fluorescence intensity (MFI) of mean ± standard deviation. Corresponding p-values ​​for significance are indicated: **p < 0.01, ***p < 0.001.

[0042] Figures 6a to 6c are results showing that APX-115 can inhibit collagen-induced p38 MAPK / cPLA2 / TXA2 production signaling. Washed platelets (5 x 10⁶ 8 ( / mL) was incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, followed by stimulation with 10 μg / mL collagen. (Figs. 6a and 6b) After 2 minutes of stimulation, platelet lysates were used for immunoblot analysis to measure the phosphorylation of p38 MAPK (Fig. 6a) and cytoplasmic phospholipase (cPLA2) (Fig. 6b). The top panel shows representative immunoblot analyses, with the locations of molecular weight (kDa) markers indicated. Quantitative data are the mean ± standard deviation of signal intensity. (Fig. 6c) After 5 minutes of stimulation, the concentration of thromboxane B2 (TXB2) in the supernatant separated from the reaction mixture was measured using an ELISA kit. All values ​​are mean ± standard deviation. The corresponding p-values ​​for significance are indicated as follows: *p < 0.05, ***p < 0.001.

[0043] Figures 7a and 7b are results showing that APX-115 can inhibit collagen-induced ERK5 activation and PS exposure. Washed platelets (5 x 10⁶ 8 ( / mL) was incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, followed by stimulation with 10 μg / mL collagen. (Fig. 7a) After 2 minutes of stimulation, platelet lysates were used for immunoblot analysis to measure ERK5 phosphorylation. The left panel shows a representative immunoblot analysis with the locations of molecular weight (kDa) markers indicated. Quantitative data are the mean ± standard deviation of signal intensity. (Fig. 7b) After 5 minutes of stimulation, platelets were incubated with annexin V-FITC, and platelet surface expression of phosphatidylserine was monitored via flow cytometry. The left panel shows a representative dot plot. Quantitative data are the mean ± standard deviation of the positive rate of annexin V binding. The corresponding p-values ​​for significance are indicated: *p ​​< 0.05, ***p < 0.001.

[0044] Figures 8a to 8c show APX-115 in collagen-induced platelets as an integrated α IIb These results show that the β3 activation signaling pathway can be inhibited. (Figs. 8a to 8c) Washed platelets (5 x 10⁶ 8 ( / mL) was stimulated with 10 μg / mL collagen after incubating with a vehicle or a specified concentration of APX-115 for 10 minutes. (Fig. 8a) After 5 minutes of stimulation, platelets were stimulated with the anti-active conjugate α labeled with FITC. IIb Platelet surface consolid α through flow cell analysis after incubation with β3 antibody (PAC1-FITC) IIbβ3 activation was monitored. The top panel shows a representative histogram. Quantitative data is the mean fluorescence intensity (MFI) as mean ± standard deviation. (Fig. 8b) After 2 minutes of stimulation, platelet lysates were used for immunoblot analysis to measure the phosphorylation of vasodilator-stimulating protein (VASP). The top panel shows a representative immunoblot analysis, with the locations of molecular weight (kDa) markers indicated. Quantitative data is the mean ± standard deviation of signal intensity. (Fig. 8c) Washed platelets (5 x 10⁶ 8 cGMP ( / mL) was incubated with a vehicle without collagen or with APX-115 (30 μM) for 5 minutes. cGMP concentrations in platelets were measured using an ELISA kit. Data are mean ± standard deviation. The corresponding p-values ​​for significance are as indicated: **p < 0.01, ***p < 0.001.

[0045] Figures 9a to 9c show the results illustrating the effects of APX-115 on platelet adhesion and thrombus formation (in vitro under flow conditions) and on arterial thrombosis and hemostasis (in vivo). (Figure 9a) Platelets labeled with DiOC6 (1 X 10⁶ 9( / mL) was incubated with a vehicle or a specified concentration of APX-115 for 10 minutes, after which it was allowed to pass through a collagen-coated surface at a constant shear rate. The left panel shows representative images of platelet adhesion and thrombus formation on the collagen. The size bar represents 100 μm. The chamber surface coverage of DiOC6 fluorescence-positive platelets was quantified. Quantitative data are the mean ± standard deviation as a percentage of the vehicle control surface coverage. (Fig. 9b) Carotid artery occlusion was induced by applying 20% ​​w / v ferric chloride 1 hour after oral administration of APX-115 (50 mg / kg body weight). Occlusion time was measured using a time-transit ultrasound flowmeter. The left panel shows a representative trace of blood flow. Quantitative data are the time to thrombotic occlusion as the mean ± standard deviation. The dot in each bar diagram represents an independent observation. The corresponding p-values ​​for significance are as indicated: **p < 0.01, ***p < 0.001. (Fig. 9c) Rat tail hemorrhage was measured 1 hour after administration of APX-115 or vehicle, as in B. Quantitative data are the mean ± standard error of hemorrhage time. The dot in each bar plot represents an independent observation. The corresponding p-values ​​for significance are as indicated: ns, p > 0.05 (not significant).

[0046] Figure 10 shows the results illustrating the effect of APX-115 on thrombin-induced platelet aggregation. Platelets purified from a healthy donor (5×10⁻⁶ 8 Platelets ( / mL) were stimulated with 0.1 U / mL thrombin after treatment with a vehicle (0.5% DMSO) or a specified concentration of APX-115 for 10 minutes. Platelet aggregation was evaluated using turbidimetry and expressed as a percentage of the change in light transmittance. The left panel shows representative aggregation traces. Quantified data are provided as mean ± standard deviation. Statistically significant p-values ​​are as follows: **p < 0.01 and ***p < 0.001.

[0047] Figures 11a and 11b show the results illustrating the effect of selective NOX inhibitors on platelet aggregation. Platelets purified from a healthy donor (5 x 10⁶ 8 ( / mL) was treated with specified concentrations of APX-115, ML171, or GSK2795039 for 10 minutes, followed by stimulation with 10 μg / mL collagen (Fig. 11a) or 0.25 U / mL thrombin (Fig. 11b). Platelet aggregation was evaluated using turbidity measurements and expressed as the percentage of maximum aggregation. Quantified data are provided as mean ± standard deviation, with n=3. Statistically significant p-values ​​are as follows: *p < 0.05 and **p < 0.01, compared to the vehicle control group. Comparisons were made using the Bonferroni modified method following one-way ANOVA for multiple comparisons. Reagent information is as follows: ML171 (Cat# 175226), Thrombin (Cat# T6884), GSK2795039 (Cat# HY-18950) (MedChem Express, Princeton, NJ, USA).

[0048]

[0049] The present invention will be described in detail below.

[0050]

[0051] In one aspect, the present invention may provide a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a compound of the following formula 1 or a pharmaceutically acceptable salt thereof:

[0052] [Chemical Formula 1]

[0053] .

[0054] The above compound acts as a pan-NOX inhibitor, blocking ROS-mediated signaling pathways by inhibiting NOX1 / 2 / 4-mediated ROS production in collagen-stimulated platelets, and ultimately inhibiting platelet activation, granule release, and aggregation. In particular, the above compound can effectively inhibit NOX1 or NOX2. By inhibiting the oxidative inactivation of PTPs by ROS, the above compound acts as follows: 1) inhibiting activation by specific tyrosine phosphorylation of Syk, LAT, Vav1, and Btk within the collagen receptor GPVI signaling pathway, thereby inhibiting PLCγ2, PKC, and Ca 2+ 1) Reduces mobilization activation; 2) inhibits ROS-induced p38 MAPK activation, thereby reducing cytoplasmic PLA2 phosphorylation and TXA2 production; 3) inhibits ROS-induced ERK5 activation, thereby reducing exposure to coagulogenic PS; 4) increases cGMP, thereby inhibiting the activation of complex αIIbβ3 and increasing the level of PKG-induced VASP phosphorylation.

[0055] In an exemplary embodiment, the compound of Formula 1 may be APX-115, but is not limited thereto.

[0056] The term “salt” means a salt according to one aspect of the present disclosure having the desirable activity of a parent compound, and may be a salt that does not cause serious irritation to an organism to which the compound is applied and does not impair the biological activity and physical properties of the compound.

[0057] The above salt refers to an addition salt of an inorganic salt, an organic salt, or a metal salt of a compound. The above inorganic salt may be a hydrochloride, bromate, phosphate, sulfate, or disulfate. The above organic salt may be a formate, acetate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl, trichloroacetate, trifluoroacetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, emvonate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate. The above metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0058] The term “thrombotic disease” refers to a disease in which a thrombus is abnormally formed within a blood vessel, obstructing or blocking the flow of blood.

[0059] The above pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents or emulsification promoters, salts and / or buffers for osmotic pressure regulation, and other therapeutically useful substances, and may be formulated into various oral or parenteral administration forms according to conventional methods.

[0060] The above oral formulations include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, powders, granules, granules, pellets, etc., and these formulations may contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycol) in addition to the active ingredient. The tablet may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and, in some cases, may contain pharmaceutical additives such as disintegrants, absorbents, coloring agents, flavoring agents, and sweeteners, such as starch, agar, alginic acid, or its sodium salt. The tablet may be manufactured by conventional mixing, granulation, or coating methods. Additionally, the parenteral agent may be in the form of a rectal, topical, subcutaneous, or transdermal administration, and may be in the form of, for example, an injection, drops, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, or patch, but is not limited thereto.

[0061] The determination of the dosage of the above compound or its salt is within the level of a person skilled in the art, and the daily dosage of the drug varies depending on various factors such as the degree of symptom progression, onset time, age, health condition, and complications of the subject to administration, but generally, based on adults, the above compound may be administered in divided doses of 1 μg / kg to 200 mg / kg, preferably 50 μg / kg to 50 mg / kg, once to three times a day, and the above dosage does not limit the scope of the present specification in any way.

[0062] In an exemplary embodiment, the compound may be a pyrazole derivative. For example, the pyrazole derivative may be 3-Phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol.

[0063] In an exemplary embodiment, the composition may inhibit the activation of platelets or the formation of blood clots.

[0064] In an exemplary embodiment, the composition may have one or more of the following features: (a) inhibition of reactive oxygen species (ROS) production in platelets by collagen stimulation, (b) inhibition of the GPVI signaling pathway, (c) inhibition of one or more tyrosine phosphorylation selected from the group consisting of Syk, LAT, Vav1, Btk, and PLCγ2, and (d) cytoplasmic calcium ions (Ca 2+ (e) reduction in concentration, (e) exposure to P-selectin or inhibition of integrin αIIbβ₃ activation, (f) inhibition of platelet adhesion to collagen.

[0065] In an exemplary embodiment, the composition can effectively inhibit the NOX1 and NOX2 pathways simultaneously.

[0066] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0067] In an exemplary embodiment, the composition may not inhibit the hemostatic action of blood. Among conventional thrombosis treatment drugs, antiplatelet agents inhibit platelet aggregation, but they are accompanied by side effects that increase the risk of bleeding by excessively inhibiting platelet function, which plays an important role in hemostasis. The compound of Formula 1 of the present invention inhibits platelet aggregation and thrombus formation without inhibiting hemostatic action, so it can reduce the risk of bleeding that may occur in patients.

[0068] In an exemplary embodiment, the thrombotic disease may be selected from the group consisting of arterial thrombosis, venous thrombosis, coronary artery thrombosis, deep vein thrombosis, cerebral artery thrombosis, peripheral vascular thrombosis, arterial embolism, venous embolism, renal embolism, chronic arterial occlusion, thrombotic microangiopathy, pulmonary infarction, cerebral infarction, cerebral embolism, and thrombophlebitis.

[0069] In another aspect, the present invention may provide a health functional food composition for improving blood circulation comprising a compound of Formula 1 or a food-grade acceptable salt thereof. The compound and the salt are as described above.

[0070] The term “blood circulation” refers to the movement of blood through blood vessels to various parts of the body. Blood supplies oxygen and nutrients to each tissue of the body and removes waste products produced by cells. In addition, blood plays a role in maintaining homeostasis within the body by transporting hormones to their sites of action, defending cells against external harmful substances, maintaining an appropriate body temperature, and performing hemostasis. Therefore, smooth blood flow is crucial for maintaining bodily functions, and impaired blood circulation can increase the risk of developing the aforementioned thrombotic diseases.

[0071] The above-mentioned health functional food composition may include food-grade acceptable food additives and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods. The formulation of the above-mentioned health functional food composition is not particularly limited, but may be formulated, for example, into tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, etc. For each formulation of the food composition, in addition to the active ingredient, ingredients commonly used in the field can be appropriately selected and combined by a person skilled in the art without difficulty according to the formulation or purpose of use, and a synergistic effect may occur when applied simultaneously with other raw materials.

[0072] The above health functional food composition may include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and promoters (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, the above food compositions may include fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not particularly important, they are generally included in the range of 0 to about 50 parts by weight per 100 parts by weight of the composition according to one embodiment.

[0073] In an exemplary embodiment, the composition may inhibit ROS generation by platelets; activation of platelets; or formation of thrombi.

[0074] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0075] In another aspect, the present invention aims to provide a cosmetic composition for improving blood circulation comprising a compound of Formula 1 or a cosmetically acceptable salt thereof. The compound, salt, and blood circulation are as described above.

[0076] The appearance of the above cosmetic composition may contain a cosmetically or dermatologically acceptable medium or base. This may be provided in any formulation suitable for topical application, for example, in the form of a solution, gel, solid, paste anhydrous product, emulsion obtained by dispersing an oil phase in an aqueous phase, suspension, microemulsion, microcapsule, microgranule, or ionic (liposome) and non-ionic vesicular dispersants, or in the form of a cream, skin, lotion, powder, ointment, spray, or concealer stick. These compositions may be prepared according to conventional methods in the art. The composition according to this specification may also be prepared in the form of a foam or in the form of an aerosol further containing a compressed propellant.

[0077] The above cosmetic composition is not particularly limited in its formulation and can be formulated into, for example, cosmetics such as softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, or body essence.

[0078] The above cosmetic composition may be a topical skin preparation composition. The above topical skin preparation is a general term that may include anything applied externally to the skin, and may include cosmetics and pharmaceuticals of various formulations.

[0079] In an exemplary embodiment, the composition may inhibit ROS generation by platelets; activation of platelets; or formation of thrombi.

[0080] In an exemplary embodiment, the composition may inhibit collagen or thrombin-induced platelet aggregation.

[0081]

[0082] Examples

[0083] 1. Experimental Materials and Methods

[0084] 1-1. Ethical Declaration

[0085] The animal experiments were reviewed and approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC No.: SNU-220331-6-5). Human blood samples were collected from healthy volunteers, and the subsequent experimental procedures were reviewed and approved by the Seoul National University Institutional Review Board. The approved protocol number is 2206 / 001-006. All human participants provided written informed consent prior to participating in the experiment.

[0086]

[0087] 1-2. Reagents and Antibodies

[0088] APX-115 (Product No.: U109969; Achemblock, Hayward, CA, USA), DCFH2 (Product No.: HY-D0940; MedChem Express, Princeton, NJ, USA), CM-H2DCFDA (Product No.: C6827), DiOC6 (Product No.: D273), Fluo-3-AM (Product No.: F1242) (All Molecular Probes, Eugene, OR, USA), AEBSF (Product No.: A-540; Gold Biotechnology, St. Louis, MO, USA), aprotinin, NaCl, NaHCO3 (Product Nos.: 0332, 0241, 0335) (All Amresco, Solon, OH, USA), CaCl2, DMSO, EGTA, FeCl3, glucose (Product Nos.: C5670, D8418, E4378, 31235, G7021), KH2PO4, Na2HPO4, Na3VO4, Na4P2O 7·10H2O, NaF (Product No.: P5655, S9763, S6508, S6422, S7920), N,N-Dimethylacetamide, Paraformaldehyde, Prostaglandin E1, Sodium Citrate (Product No.: D5511, P6148, P5515, S4641), Triton X-100, Tween 80, β-glycerophosphate (Product No.: T9284, P4780, G9422) (All Sigma-Aldrich, St. Louis, MO, USA), Collagen (Product No.: 385; Chrono-Log, Havertown, PA, USA), Citric Acid (Product No.: 4337; Duksan, Seoul, South Korea), EDTA, KCl, leupeptin, MgCl2 (Product No.: 75829, 74075, 78436, 75826) (all from USB, Cleveland, OH, USA), FITC-labeled annexin V (product number: 556419; BD Biosciences, San Jose, CA, USA), and HEPES (product number: 11344041; Thermo Fisher Scientific, Waltham, MA, USA) were purchased from each company.

[0089] The following antibodies were used: anti-phospho-Btk (Tyr 551 ) Antibody (Product No.: 44-1355G, Invitrogen, New York, NY, USA), anti-Btk (Product No.: sc-81238), anti-LAT (Product No.: sc-365626), anti-phospho-PLCγ2 (Tyr 753 ) (Product No.: sc-130252), anti-PLCγ2 (Product No.: sc-407), anti-Syk (Product No.: sc-1240), anti-VASP (Product No.: sc-46669), anti-phospho-Vav1 (Tyr 174) (Product No.: sc-101858) Antibodies (All Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-cPLA2 (Ser 505 ) (Product No.: 2831), anti-cPLA2 (Product No.: 2832), anti-phospho-ERK5 (Thr 218 / Tyr 220 ) (Product No.: 3371), anti-ERK5 (Product No.: 3372), anti-phospho-p38 MAPK (Thr 180 / Tyr 182 ) (Product No.: 9211), anti-p38 MAPK (Product No.: 8690), anti-phospho-Syk (Tyr525 / 526) (Product No.: 2711), anti-phospho-VASP (Ser 239 ) (Product No.: 3114) Antibodies (All Cell Signaling Technology, Danvers, MA, USA), anti-phospho-LAT (Tyr 200 ) (Product No.: ab68139), anti-Vav1 antibody (Product No.: ab40875, both Abcam, Cambridge, UK), anti-phosphotyrosine (4G10) antibody (Product No.: 05-321) Sigma-Aldrich, St. Louis, MO, USA), anti-β-actin antibody (Product No.: LF-PA0207) (Abfrontier, Seoul, South Korea), goat anti-rabbit IgG and anti-mouse IgG labeled with horseradish peroxidase (Product Nos.: 5220-0336, 5450-0011; both Seracare Life Sciences, Milford, MA, USA), PAC1-FITC (Product No.: 340507) and CD62P-PE (Product No.: 555524) antibodies (both BD Biosciences, San Jose, CA, USA).

[0090]

[0091] 1-3. Preparation of Human Platelets

[0092] Blood was collected from healthy, drug-free volunteers by venipuncture and placed in tubes containing an anticoagulant acid / citric acid / dextrose solution (Product No.: 364816) (Becton Dickson, Franklin, NJ, USA). Platelet-rich plasma (PRP) was centrifuged at 150Xg for 15 minutes, followed by an additional centrifugation at 300Xg for 10 minutes to concentrate the platelets. The platelet pellet was suspended in a solution containing Tyrode's-HEPES buffer (10 mM HEPES [pH 7.4], 129 mM NaCl, 0.8 mM KH2PO4, 8.9 mM NaHCO3, 2.8 mM KCl, 0.8 mM MgCl2, 5.6 mM glucose), 2 mM EDTA, 10% citric acid / citric acid / dextrose solution, and 1 μM prostaglandin E1, and then centrifuged again. The supernatant was discarded, and the platelet pellet was resuspended in Tyrode's-HEPES buffer to the desired concentration. Unless otherwise specified, washed platelets were treated with 1 mM CaCl2 for 2 minutes prior to stimulation.

[0093]

[0094] 1-4. Light Transmission Aggregometry

[0095] Platelet aggregation was measured in silicone-treated glass cuvettes using a 4-channel aggregator (Chrono-Log, Havertown, PA, USA) at 37 °C with continuous stirring at 1,000 rpm. Data were collected using Aggrolink software (model number: 700; Chrono-Log, Havertown, PA, USA).

[0096]

[0097] 1-5. Measurement of Intracellular ROS and Cytoplasmic Ca2+ Concentrations

[0098] Intracellular ROS and Ca2+ concentrations were measured using CM-H2DCFDA and Fluo-3-AM, respectively. Washed platelets (5 × 10⁻⁶ 8 / mL) was incubated in PBS containing 5 μM CM-H2DCFDA or 1 μM Fluo-3-AM for 30 minutes at 37°C in a dark place, then centrifuged and resuspended in Tyrode's-HEPES buffer. Probe-treated platelets (5 × 10 8 ( / mL) was stimulated in a fluorescence spectrophotometer cuvette with continuous stirring at 37 °C and 1,000 rpm. Fluorescence of CM-DCF (absorption at 495 nm / emission at 525 nm) or Fluo-3 (absorption at 488 nm / emission at 525 nm) was measured using an FP-8350 model (Jasco, Tokyo, Japan) fluorescence spectrophotometer. The initial signal was recorded for 20 seconds prior to collagen stimulation and used as a baseline, after which measurements were continued. For each sample, the fluorescence signal was normalized to the baseline.

[0099]

[0100] 1-6. Measurement of Extracellular ROS Concentration

[0101] Washed platelets (5 x 10 8 1 μM DCFH2 ( / mL) was added to Tyrode's-HEPES buffer and stimulated in a fluorescence spectrophotometer cuvette with continuous stirring at 800 rpm at 37 °C. The fluorescence of DCF (absorption at 488 nm / emission at 525 nm) was measured using a fluorescence spectrophotometer as described above.

[0102]

[0103] 1-7. Western Blotting

[0104] After stimulating with a thermomixer at 37 °C and 1,000 rpm with continuous stirring, platelets were lysed in cell extraction buffer (20 mM HEPES [pH 7.0], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 2 mM EGTA, 20 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg / mL leupeptin, 1 μg / mL aprotinin, 1 mM AEBSF). Cell debris was removed by centrifugation at 12,500 Xg for 10 minutes. Analysis with specific antibodies was performed using equal volumes of protein-adjusted cell lysates. Band intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA).

[0105]

[0106] 1-8. TXB2 ELISA

[0107] After treatment using a thermomixer at 37 °C with continuous stirring at 1,000 rpm, the reaction was stopped by adding 5 mM EDTA and 200 μM Indomethacin. Platelet reaction mixture (5 × 10 8 The platelets (1 / mL) were immediately centrifuged for 30 seconds to obtain the supernatant. The concentration of TXB2 in the supernatant was measured using a Cytation3 microplate spectrophotometer (BioTek, Burlington, VT, USA) with an ELISA kit (Cat# 501020; Cayman, Ann Arbor, MI, USA).

[0108]

[0109] 1-9. Flow Cytometry and Analysis

[0110] Fluorescently labeled antibodies CD62P-PE (0.5 μg / mL), PAC1-FITC (0.5 μg / mL), or annexin V-FITC (0.1 mg / mL) were added to stain surface P-selectin, activated conjugate αIIbβ3, and PS exposure, respectively. Platelet mixtures were stimulated with collagen at 37°C for 5 minutes in the dark. Each sample was fixed with 1% paraformaldehyde and diluted with PBS. All analyses were performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) with a minimum of 5 x 10⁶ 4 Canine cells were used as samples. Platelet populations were identified and gated based on forward scattering and lateral scattering characteristics. Data were analyzed using FlowJo software 10.9 (FlowJo LLC, Ashland, OR, USA).

[0111]

[0112] 1-10. Measurement of ATP Release

[0113] Platelets were incubated in a white, flat-bottomed 96-well plate and then stimulated with collagen. Luciferin-luciferase reagent from the ATP bioluminescence measurement kit (ab113849; Abcam, Cambridge, CB2 0AX, UK) was added to each well, and the 96-well plate was placed in an LB 960 Centro microplate luminometer (Berthold, Bad Wildbad, Germany). After shaking the samples according to an automated protocol, luminescence was measured and recorded for 5 minutes at 30-second intervals.

[0114]

[0115] 1-11. cGMP ELISA

[0116] Platelet reaction mixture (5 x 10 8Platelets (1 / mL) were centrifuged at 10,000Xg for 30 seconds using a 0.8 μm centrifugal filter (Cat# VK01P042; Sartorius, Elk Grove, IL, USA) to selectively remove the supernatant. Platelets remaining on the filter membrane were lysed in 0.1 M HCl containing 1% Triton X-100. The lysed sample was centrifuged at 12,500Xg for 10 minutes to remove debris. The resulting supernatant was then used for analysis. The cGMP concentration in the supernatant was measured using a Cytation3 microplate spectrophotometer with an ELISA kit (Cat# ADI-900-164; Enzo Life Sciences, Exeter, UK).

[0117]

[0118] 1-12. Measurement of Platelet Adhesion and Thrombus Formation under Flow Conditions

[0119] Washed platelets (1 x 10 9 ( / mL) was incubated in 1 μM DiOC6 at 37 °C for 10 minutes. Collagen-coated coverslips (Cat# GG-12-Collagen; Neuvitro, Camas, WA, USA) were mounted in a parallel-plate flow chamber (Chamlide CF, Live Cell Instrument, Seoul, Korea). Platelets stained with DiOC6 were 1000 s-1The sample was passed through the collagen matrix at a rate using a pump injector (Harvard Apparatus, Holliston, MA, USA) until 1 mL of the sample was depleted. Non-attached platelets remaining in the chamber were washed with PBS. Platelet thrombi and attached platelets were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and visualized using a fluorescence microscope (Ts2-FL, Nikon, Tokyo, Japan). Representative images were taken in 5 to 10 different fields. Flow chamber surface coverage was calculated using ImageJ software (NIH, Bethesda, MD, USA).

[0120]

[0121] 1-13. Evaluation of Arterial Thrombosis

[0122] Mice (C57BL / 6 J, male) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and reared at the Specified Pathogen-Free Animal Facility of Seoul National University. Mice were fed a standard diet (Cat# 2018; Harlan Teklad, Madison, WI, USA) on a free basis. Male mice aged 10–11 weeks were divided into three groups. Mice were orally administered APX-115 at a dose of 10 mL / kg body weight, dissolved in 10% N, N-dimethylacetamide, 10% Tween 80, and 80% distilled deionized water, as previously described [Joo, JH et al. A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase. Sci Rep 6:22389; 2016]. After anesthesia via peritoneal injection containing 100 mg / kg alpaxarone (Jurox, Cat# 520400; New South Wales, Australia) and 10 mg / kg xylazine (Rompun; Bayer Korea, Seoul, Korea), the right carotid artery was exposed. Carotid artery occlusion was performed according to the previously described method [Bonnard, T.; Hagemeyer, CE Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel.J Vis Exp:e52838; 2015.]. Carotid artery occlusion was measured by recording blood flow using a Perivascular Flowmeter (Model TS420, Transonic Systems Inc., Ithaca, NY, USA). A Perivascular Flowprobe (Model MA0.5PSB, Transonic Systems Inc.) was placed in the carotid artery, separated through an incision, and 0.3–0.The blood flow was adjusted to maintain a baseline of 9 mL / s. Vascular injury was induced 1 hour after APX-115 administration by applying a 1 x 1 mm filter paper saturated with 20% FeCl3 near the carotid artery. After 3 minutes, the filter paper was removed and blood flow was measured. Initial and final data points were collected at 0.5-second intervals, and the time taken to reach stable occlusion was calculated. Occlusion was deemed stable when blood flow decreased to 0 mL / s for at least 30 seconds.

[0123]

[0124] 1-14. Evaluation of tail hemorrhage time

[0125] Mice (C57BL / 6 J, male) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and anesthetized with APX-115 for the arterial thrombosis measurement method described above. The tail was cut to a fixed diameter of 1.5 mm, and the remaining portion was immersed in an isotonic solution preheated to 37 °C. Hemorrhage was visually tracked. The time until stable cessation of bleeding was recorded, and bleeding was considered stopped if it did not recur within 1 minute after cessation.

[0126]

[0127] 1-15. Statistical Analysis

[0128] Means and standard deviations were calculated for all experimental groups. In all experiments, n represents the number of independent experiments. Data were analyzed using Student's t-test or Mann-Whitney U test for two-group comparisons and Dunnett's test following one-way ANOVA for multiple-group comparisons. Data analysis was performed using GraphPad PRISM software 10 (GraphPad Software Inc., La Jolla, CA, USA). A P-value of ≤0.05 was considered statistically significant.

[0129]

[0130] 2. Experimental Results

[0131] 2-1. APX-115 inhibits collagen-induced aggregation and ROS production in platelets.

[0132] The concentration of APX-115 used in this experiment was selected based on prior research and experimental verification. Through dose-response experiments, it was confirmed that APX-115 inhibits collagen-stimulated aggregation of human platelets at an IC50 of approximately 13.5 μM (Fig. 2a). Based on these results, the concentration of APX-115 in this experiment was verified as a suitable concentration for studying the effect on collagen-stimulated platelet aggregation.

[0133] It is known that NOX1 and NOX2 deficiencies inhibit thrombin-induced aggregation in mouse platelets. Therefore, the inventors investigated the effect of APX-115 on human platelet aggregation induced by thrombin. APX-115 also significantly inhibited thrombin-induced platelet aggregation (Fig. 10). These results suggest that APX-115 can inhibit GPVI and GPCR-mediated platelet activation. However, considering the fact that ROS production in human platelets induced by collagen stimulation increases to a much higher level than when stimulated with GPCR ligands such as thrombin, ADP, and TXA2, the inventors further investigated the effect of APX-115 on collagen-induced platelet activation and function.

[0134] To evaluate the effect of APX-115 on the collagen-induced elevation of ROS levels in platelets, the redox-sensitive fluorescent probe 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was used (Fig. 2b). Collagen stimulation significantly increased ROS production in human platelets. APX-115 reduced the collagen-induced elevation of ROS in platelets in a concentration-dependent manner. The effect of APX-115 on the collagen-induced elevation of extracellular ROS levels was also investigated using 2',7'-dichlorodihydrofluorescein (DCFH2), a redox-sensitive and membrane-impermeable probe (Fig. 2c). It was confirmed that extracellular ROS production significantly increased due to collagen stimulation. APX-115 prevented the collagen-induced elevation of extracellular ROS in a concentration-dependent manner. These results suggest that APX-115 may have an antiplatelet effect by blocking NOX-mediated ROS production.

[0135]

[0136] 2-2. APX-115 attenuates collagen-stimulated GPVI signals in platelets.

[0137] Collagen-activated GPVI promotes the phosphorylation of T cell activation linkage (LAT) tyrosine residues by splenic tyrosine kinase (Syk), which induces the formation of LAT signaling complexes along with important components such as Vav1, Bruton's tyrosine kinase (Btk), and PLCγ2. Protein tyrosine phosphorylation is a key feature of GPVI-stimulated signaling. Oxidative inactivation of protein tyrosine phosphatases (PTPs) by ROS can amplify the GPVI signaling pathway in collagen-stimulated platelets. Based on the finding that APX-115 inhibited ROS production in collagen-stimulated platelets, the inventors investigated the effect on protein tyrosine phosphorylation. The level of total tyrosine phosphorylation induced by collagen was significantly increased compared to the baseline value (Fig. 3a). Protein tyrosine phosphorylation was inhibited when platelets were pretreated with APX-115 (30 μM) and then stimulated by collagen. This suggests that APX-115 can prevent the oxidative inactivation of PTPs by blocking NOX-dependent ROS generation. The binding of Syk, Vav1, Btk, and PLCγ2 within the LAT signaling complex is primarily mediated by a binding module between phosphorylated tyrosine residues and the SH2 domain, ultimately inducing PLCγ2 activation. To determine whether APX-115 regulates the activation of the GPVI signaling pathway, the inventors evaluated the tyrosine phosphorylation-based activation of Syk, LAT, Vav1, and Btk. Western blot analysis using phosphorylation-specific antibodies indicated that collagen stimulation increased phosphorylation at Tyr525 / Tyr526 in Syk, Tyr200 in LAT, Tyr174 in Vav1, and Tyr551 in Btk (Fig. 3b). The activation of each signaling protein depends on the tyrosine phosphorylation of the corresponding protein, which can also be used to measure the activity of individual molecules.After treatment with APX-115, the activation of Syk, LAT, Vav1, and Btk in collagen-stimulated platelets was selectively inhibited by reducing tyrosine phosphorylation. These results suggest that APX-115 can protect PTPs from oxidative inactivation by blocking NOX-mediated ROS production, thereby inhibiting the activation of Syk, LAT, Vav1, and Btk.

[0138]

[0139] 2-3. APX-115 inhibits collagen-induced PLCγ2 activation and Ca2+ migration in platelets.

[0140] In response to collagen stimulation, the phosphorylation of Tyr753, a downstream target of PLCγ2, is associated with the tyrosine phosphorylation-based activation of Vav1 and Btk, which leads to increased activation of PLCγ2. APX-115 blocked important upstream molecules, allowing for the investigation of Tyr753 phosphorylation of PLCγ2 in collagen-stimulated platelets. As shown in Figure 4a, APX-115 prevented collagen-induced Tyr753 phosphorylation of PLCγ2. In addition, the inventors' experimental results show that APX-115 reduces PLCγ2 activity by blocking the activation of Syk, LAT, Vav1, and Btk, thereby preventing NOX-mediated ROS production. When PLCγ2 generates diacylglycerol and inositol-1,4,5-trisphosphate (IP3) in collagen-stimulated platelets, PKC is activated, increasing cytoplasmic Ca2+ concentration and causing Ca2+ to move from intracellular depots. Therefore, the inventors measured cytoplasmic Ca2+ concentration using Fluo-3-AM as a probe. Since cytoplasmic Ca2+ concentration rises as Ca2+ is released from internal depots in the absence of external Ca2+, cytoplasmic Ca2+ concentration was evaluated in the presence of 0.5 mM EGTA (Fig. 4b). APX-115 effectively inhibited collagen-induced Ca2+ movement from internal depots. Furthermore, APX-115 successfully prevented collagen-induced cytoplasmic Ca2+ elevation even under conditions where external Ca2+ was present (Fig. 4c). These results suggest that the antiplatelet action of APX-115 may influence the regulation of Ca2+ movement and PLCγ2 activation.

[0141]

[0142] 2-4. APX-115 inhibits collagen-induced platelet granule release.

[0143] After platelets are activated, the release of active substances from dense granules and alpha-granules plays an important role in enhancing platelet activation. Granule release in collagen-stimulated platelets is promoted by PKC activation induced by PLCγ2 and an increase in cytoplasmic Ca2+. The inventors evaluated the effect of APX-115 on collagen-induced granule release as it inhibits PLCγ2. Platelet dense granule release was evaluated through ATP release analysis. Figure 5a shows that APX-115 significantly reduces the amount of ATP released from collagen-stimulated human platelets. P-selectin acts as an adhesion molecule that promotes platelet-leukocyte contact on the platelet surface where alpha-granules have degranulated after platelet activation. In a flow cell analysis using a PE-labeled anti-P-selectin antibody (CD62P-PE), collagen increased P-selectin expression on the platelet surface, which was strongly inhibited by APX-115 (Fig. 5b).

[0144]

[0145] 2-5. APX-115 attenuates collagen-induced p38 MAPK / cPLA2 / TXA2 generation signaling activation in platelets.

[0146] The collagen-induced platelet aggregation process is amplified by TXA2, an agonist of the secondary wave. Cytoplasmic phospholipase (cPLA2) is an important enzyme that aids in TXA2 production in collagen-stimulated platelets, releases arachidonic acid from cell membrane phospholipids, and its activity is increased by p38 MAPK-dependent Ser505 phosphorylation. It is known that the activation of p38 MAPK is inhibited in GPVI-stimulated platelets by selective NOX2 inhibitors or by a deficiency of p47phox, the regulatory subunit of NOX2. Furthermore, prior studies have shown that appropriate p38 MAPK activation and subsequent TXA2 synthesis in GPVI-stimulated platelets depend on ROS generated from NOX1. Accordingly, the inventors investigated whether APX-115 regulates the cPLA2 activation of p38 MAPK. Collagen-induced platelet activation results in Thr180 / Tyr182 phosphorylation of p38 MAPK and Ser510 phosphorylation of its downstream protein, cPLA2. When pretreated with APX-115, these phosphorylations were significantly reduced (Figs. 6a and 6b). Next, the inventors evaluated the effect of APX-115 on the production of TXB2, a stable metabolite of TXA2. TXB2 production in collagen-stimulated platelets was significantly inhibited by APX-115 (Fig. 6c). These results suggest that the antiplatelet action of APX-115 is partly attributed to the downregulation of the ROS-dependent p38 MAPK / cPLA2 / TXA2 production signaling pathway.

[0147]

[0148] 2-6. APX-115 inhibits collagen-induced ERK5 activation and PS exposure in platelets.

[0149] The redox-sensitive MAP kinase ERK5 is stimulated by NOX-mediated ROS in activated platelets. This contributes to fibrin production by externalizing phosphatidylserine (PS), a coagulation-promoting phospholipid. Therefore, the inventors investigated the effect of APX-115 on the ability of ERK5 to promote increased PS externalization. Thr218 / Tyr220 phosphorylation of ERK5 in collagen-stimulated platelets was significantly inhibited upon pretreatment with APX-115 (30 μM) (Fig. 7a). Next, the effect of APX-115 on platelet coagulation responses was evaluated by measuring annexin V-FITC, which binds to platelets, as an indicator of PS exposure using flow cytometry. Although PS exposure significantly increased upon collagen treatment, the proportion of annexin V-positive platelets decreased upon APX-115 pretreatment (Fig. 7b). These results support the hypothesis that APX-115 may contribute to regulating the coagulation-promoting state in thrombotic diseases by blocking ERK5 activation through its ability to reduce ROS production.

[0150]

[0151] 2-7. APX-115 inhibits the collagen-induced signaling pathway for platelet integrin αIIbβ3 activation.

[0152] Platelet adhesion and aggregation are significantly influenced by the internal-external activation of integrin αIIbβ3, which leads to binding with high affinity to fibrinogen. Considering that NOX-mediated ROS plays a significant role in integrin αIIbβ3 activation in GPVI-stimulated platelets, the inventors evaluated the effect of APX-115 on αIIbβ3 internal-external signaling. Flow cytometry was used to determine whether APX-115 inhibits collagen-induced activation modification of αIIbβ3 using an FITC-labeled activating integrin αIIbβ3 antibody (PAC1-FITC) (Fig. 8a). Vasodilator-stimulated phosphorylation protein (VASP), an adaptive molecule that regulates integrin αIIbβ3 activation, plays a crucial role in preventing platelet activation. Triple NOX-deficient mouse platelets consistently exhibited higher intracellular cGMP levels under resting conditions, and under collagen-stimulated conditions, the phosphorylation level of VASP Ser239 was significantly higher. Furthermore, it was revealed that ROS generated from NOX block the cGMP / PKG signaling pathway in platelets. Therefore, the effects of APX-115 on VASP phosphorylation and intraplatelet cGMP levels were evaluated. As shown in Figure 8b, phosphorylation of VASP Ser239 was significantly reduced after collagen stimulation, and phosphorylation decreased noticeably upon the addition of APX-115. Additionally, administration of APX-115 at a concentration of 30 μM under normal conditions resulted in a 3.7-fold increase in intraplatelet cGMP levels (Figure 8c). These results suggest that the regulation of the ROS-controlled cGMP / PKG / VASP / integrin αIIbβ3 signaling pathway contributes to the antiplatelet activity of APX-115.

[0153]

[0154] 2-8. Effects of APX-115 on Platelet Adhesion and Thrombus Formation under Flow Conditions and Confirmation of In vivo Arterial Thrombosis and Hemostatic Effects

[0155] The primary cause of platelet adhesion and aggregation is exposure to underlying cytoplasmic collagen at sites of endothelial damage. Platelets from NOX1 or NOX2-deficient mice showed reduced thrombus volume after attaching to collagen under shear force. Therefore, the inventors investigated platelet adhesion and thrombus formation under shear force in a collagen-covered flow chamber. In APX-115-treated platelets, both stable platelet adhesion and the diameter of the thrombus on the immobilized collagen were reduced (Fig. 9a). These results demonstrate that APX-115 prevents thrombus growth and inhibits stable platelet adhesion under shear force. Finally, the effect of the platelet activation inhibitory effect of APX-115 on thrombosis in vivo was evaluated. Sensitivity to thrombosis was investigated using an arterial thrombosis model by inducing carotid artery occlusion with FeCl3. As can be seen in Fig. 9b, mice administered with APX-115 showed a significant protective effect against carotid artery occlusion compared to the vehicle control group (10.6 ± 1.5 min vs. 15.9 ± 2.2 min, n = 6, p = 0.0022). The inventors also conducted a tail bleeding time experiment to evaluate the effect of APX-115 on hemostasis. As shown in Fig. 9c, APX-115 did not have a significant effect on bleeding time (201.2 ± 48.1 sec vs. 197.0 ± 22.2 sec, n = 6, p = 0.9389). This suggests that APX-115 can inhibit thrombosis without interfering with normal hemostasis.

[0156]

[0157] 2-9. Confirmation of the Inhibitory Effect of APX-115 on Collagen and Thrombin-Induced Platelet Aggregation Compared to Other NOX Inhibitors

[0158] In a comparative experiment between ML171 (NOX1-specific inhibitor) and GSK2795039 (NOX2-specific inhibitor), APX-115 demonstrated superior inhibitory effects against both stimuli (collagen and thrombin). This result clearly demonstrates the multi-NOX isoform inhibitory properties of APX-115. Specifically, ML171 effectively inhibited collagen-induced platelet aggregation but showed limited effects on thrombin, whereas GSK2795039 was relatively weak in inhibiting thrombin-induced activation. In contrast, APX-115 demonstrated broad efficacy by effectively inhibiting both the NOX1 and NOX2 pathways in both collagen and thrombin. APX-115 exhibited broader inhibition compared to the isoform-specific inhibitors ML171 (NOX1) and GSK2795039 (NOX2) (Figs. 11a, 11b). ML171 strongly inhibited collagen-induced aggregation, whereas GSK2795039 was less effective. APX-115 demonstrated superior inhibition of both collagen and thrombin-induced aggregation, highlighting its pan-NOX inhibitory activity and broader therapeutic potential.

[0159]

[0160] [National R&D projects that supported this invention]

[0161] [Project ID] 2340002209

[0162] [Project No.] NRF-2022R1A6A1A03046247

[0163] [Ministry Name] Ministry of Education

[0164] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0165] [Research Project Name] University Key Research Institute Support Project

[0166] [Research Project Title] Seoul National University Institute of Comprehensive Pharmacy

[0167] [Name of Project Performing Organization] Seoul National University Industry-Academic Cooperation Foundation

[0168] [Research Period] 2022.06.01 ~ 2025.02.28

Claims

1. A pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising a compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] .

2. In Paragraph 1, A composition in which the above compound is a pyrazole derivative.

3. In Paragraph 1, The above composition is a composition that inhibits the activation of platelets or the formation of blood clots.

4. In Paragraph 1, The above composition has one or more of the following features: (a) Inhibition of ROS (reactive oxygen species) production by platelets induced by collagen stimulation, (b) Suppression of GPVI signal transmission path, (c) One or more tyrosine phosphorylation inhibitors selected from the group consisting of Syk, LAT, Vav1, Btk and PLCγ2, (d) Cytoplasmic calcium ions (Ca 2+ ) Decrease in concentration, (e) Exposure to P-selectin or inhibition of integrin αIIbβ₃ activation, (f) Inhibition of platelet attachment to collagen.

5. In Paragraph 1, The above composition is a composition that inhibits collagen or thrombin-induced platelet aggregation.

6. In Paragraph 1, The above composition is a composition that does not inhibit the hemostatic action of blood.

7. In Paragraph 1, The above-mentioned thrombotic disease is a composition selected from the group consisting of arterial thrombosis, venous thrombosis, coronary artery thrombosis, deep vein thrombosis, cerebral artery thrombosis, peripheral vascular thrombosis, arterial embolism, venous embolism, renal embolism, chronic arterial occlusion, thrombotic microangiopathy, pulmonary infarction, cerebral infarction, cerebral embolism, and thrombophlebitis.

8. A health functional food composition for improving blood circulation comprising a compound of Chemical Formula 1 below or a food-grade acceptable salt thereof: [Chemical Formula 1] .

9. In Paragraph 8, The above composition inhibits ROS generation in platelets; activation of platelets; or formation of thrombi.

10. In Paragraph 8, The above composition is a composition that inhibits collagen or thrombin-induced platelet aggregation.

11. A cosmetic composition for improving blood circulation comprising a compound of the following chemical formula 1 or a cosmetically acceptable salt thereof: [Chemical Formula 1] .

12. In Paragraph 11, The above composition inhibits ROS generation in platelets; activation of platelets; or formation of thrombi.

13. In Paragraph 11, The above composition is a composition that inhibits collagen or thrombin-induced platelet aggregation.

Images