Use of veronicastrum stenostachyum on preparation of drugs for preventing or treating thromboembolic diseases

Abstract

The present application relates to a new use of Veronica longifolia quinone A, and in particular to the use of Veronica longifolia quinone A in the preparation of a drug for preventing or treating thromboembolic diseases. The present application finds that Veronica longifolia quinone A can significantly inhibit the platelet aggregation activity induced by different inducers in vitro: in the thrombus model experiment in vivo, the Veronica longifolia quinone A of the present application can well inhibit the FeCl3-induced carotid artery thrombosis in mice. At the same time, it is found that the Veronica longifolia quinone A of the present application does not have obvious bleeding tendency. It is shown that Veronica longifolia quinone A is expected to play a certain positive role in the prevention or treatment of thromboembolic diseases.

Description

Technical Field

[0001] This invention relates to a novel use of Veronica persica quinone A, specifically to the use of Veronica persica quinone A in the preparation of drugs for the prevention or treatment of thromboembolic diseases. Background Technology

[0002] Thromboembolic diseases seriously affect human health worldwide and have become a major health concern globally. Their development involves two stages: thrombus formation and thromboembolism. The initial stage of thrombus formation can be classified into four types: white thrombi (platelet thrombi), mixed thrombi, red thrombi, and hyaline thrombi. Platelet thrombi are the first step in thrombus formation; therefore, antiplatelet therapy is the cornerstone of thromboembolic disease treatment. Platelet thrombus formation is a multi-step process. Under normal circumstances, platelets do not interact with the intact blood vessel wall. However, upon tissue trauma, platelets adhere to the extracellular matrix through the coordinated action of ligands from platelet surface receptors such as von Willebrand factor (vWF), collagen, laminin, glycoprotein Ibα (GP Ibα), and platelet-reactive proteins, leading to initial tethering and rolling of platelets on the damaged blood vessel wall. The final step in platelet adhesion requires integrin αIIbβ3 to transition from a low-affinity state to a high-affinity state, mediating the final firm adhesion of platelets through binding to vWF and fibrinogen. Platelet adhesion triggers a signaling cascade mediated by tyrosine kinases and G protein-coupled receptors, which guides the complete activation of platelets and the accompanying particle release, leading to the recruitment and activation of other platelets. Platelet adhesion and activation result in platelet aggregation and the appearance of a procoagulant surface, promoting the formation of fibrin-rich thrombi at the site of injury. Currently used antiplatelet drugs in clinical practice, such as aspirin, clopidogrel, and ticagrelor, directly and irreversibly inhibit platelet activation and aggregation by inhibiting platelet cyclooxygenase (COX)-1 and P2Y12 receptors, exhibiting significant therapeutic effects in inhibiting thrombus formation, but also carrying a high risk of bleeding. Therefore, finding a way to inhibit thrombus formation without affecting bleeding is crucial.

[0003] Protein-protein interactions (PPIs) are fundamental units of life, playing a central role in the physiological and pathological processes of all organisms. Dysfunction of PPIs is the basis of many diseases, including thrombosis, cancer, and immune disorders; therefore, PPIs are highly direct and effective drug targets. In the application of drugs targeting platelet disorders, since PPIs do not directly inhibit platelet-activating receptors or coagulation factors, finding small-molecule inhibitors of traditional Chinese medicine that act on platelet PPIs offers a lower risk of bleeding and greater safety.

[0004] Protein disulfide isomerases (PDIs) play a crucial role in thrombosis. PDIs mediate a conformational change in integrin αIIbβ3 by binding to activated platelets, causing a transition from a low-affinity to a high-affinity state, enhancing its binding affinity to fibrinogen and promoting thrombus formation. Integrin αIIbβ3 plays a vital role in the signaling of platelet activation and aggregation; directly inhibiting its activity can lead to a significant bleeding risk. Therefore, targeting extracellular PDIs may be a therapeutic strategy for thrombotic diseases. Theoretically, because PDIs do not directly block platelet activation receptors or coagulation factors, they offer a low bleeding risk and high safety profile. Researchers have conducted numerous studies on the discovery of PDI inhibitors and the development of PDI monoclonal antibodies. Currently discovered PDI inhibitors are mostly irreversible, such as CxxC peptides, styrax peptides, P1, and TA. In the regulation of thrombotic diseases, reversible inhibitors help maintain the dynamic balance between anticoagulation and coagulation functions, making reversible PDI inhibitors a better choice. Reversible PDI inhibitors with antithrombotic effects reported in the literature are mainly flavonoids, such as rutin, quercetin, and myricetin. Among them, rutin is the most effective PDI inhibitor identified through high-throughput screening from a small molecule compound library. Intravenous injection of rutin can reduce the volume of arterial thrombi caused by laser damage without increasing bleeding time. However, some studies have shown that rutin is a non-specific inhibitor. During treatment with PDI monoclonal antibodies, Jaegyung et al. found that PDI monoclonal antibodies prolonged tail bleeding time and increased blood loss in mice, raising concerns that selective PDI inhibitors may interfere with hemostasis. Furthermore, cell-permeable molecular inhibitors may cause serious side effects because PDIs play an indispensable role in protein folding and cell viability in the endoplasmic reticulum. Other studies have shown that PDI expression precedes platelet aggregation, and that PDI binds poorly to inactive platelets, with the degree of binding increasing with platelet activation. It remains unclear how PDI activity is regulated in thrombosis; therefore, we sought to identify molecules that interact with PDI and may regulate its activity during platelet activation.

[0005] Previous literature has reported that HSP47 is expressed on the surface of human platelets, and its expression increases by 48% after GPVI activation, enhancing platelet-collagen interaction. A recently published study confirmed that downregulation of heat shock protein 47 (HSP47) on the platelet surface has an antithrombotic effect. Based on mass spectrometry proteomics, the article identified several differentially expressed proteins in the platelets of hibernating brown bears, among which HSP47 was downregulated by 55-fold. Following HSP47 downregulation, the activation of platelet activation-related receptors such as integrin αIIbβ3 and glycoprotein VI was significantly downregulated. In addition, HSP47 downregulation weakened immune cell activation and the formation of extracellular traps in neutrophils, inhibiting thrombus formation in hibernating brown bears, mice, and patients with spinal cord injuries. Previous studies have shown that HSP47 released after platelet activation binds to the platelet surface, enhancing the binding function of platelet surface molecular ligands and promoting thrombus formation, but its mechanism of action has not been reported in the literature. Searching the String protein interaction database and performing molecular docking experiments revealed a potential direct interaction between HSP47 and PDI. Immunoprecipitation experiments showed that HSP47 can form a molecular complex with PDI. Platelet aggregation experiments indicated that increasing the concentrations of both HSP47 and PDI in vitro promoted platelet aggregation. These experimental results demonstrate the interaction between platelet HSP47 and PDI, and its promotion of platelet aggregation. Since small-molecule inhibitors of traditional Chinese medicine that target platelet PPIs have lower bleeding risk and safety, this invention focuses on identifying small molecules that inhibit the interaction between platelet HSP47 and PDI.

[0006] Danshen, a traditional Chinese medicine, is the dried root or rhizome of *Salvia miltiorrhiza* Bge., a plant in the Lamiaceae family. It is slightly cold in nature, bitter in taste, and enters the Heart and Liver meridians. It is a key herb in Traditional Chinese Medicine for promoting blood circulation and removing blood stasis. Danshen contains various components, including lipophilic terpenes, water-soluble phenolic acids, fatty acids, lactones, polysaccharides, flavonoids, and quinones. Sibiriquinone A, obtained from Danshen through ligand affinity ligand binding with proteins HSP47 and PDI, is a paraquinone compound. Literature studies have shown that Sibiriquinone A has a strong inhibitory effect on lymphocyte transformation; however, other pharmacological effects are rarely reported, particularly its effects on platelet function and thrombotic diseases. Summary of the Invention

[0007] Purpose of the invention: One of the purposes of this invention is to provide a new use for Veronica persica paraquinone A in the preparation of antiplatelet aggregation and antithrombotic drugs, so as to expand the application scope of Veronica persica paraquinone A.

[0008] Technical solution:

[0009] The application of Veronica perforata quinone A in the preparation of a heat shock protein 47 / protein disulfide isomerase (HSP47 / PDI) interaction inhibitor, wherein the chemical structural formula of Veronica perforata quinone A is as follows:

[0010]

[0011] 8,8-dimethy1-2-propan-2-y1-7H-phenanthrene-1,4-dione.

[0012] The use of Veronica persica quinone A in the preparation of drugs for the treatment or prevention of thrombosis.

[0013] Application of Veronica persica quinone A in the preparation of drugs for treating or preventing platelet aggregation.

[0014] Application of Veronica persica quinone A in the preparation of drugs for the prevention or treatment of thromboembolic diseases.

[0015] Furthermore, when used as an antiplatelet aggregation drug, the final concentration of Veronica persica quinone A in the system is 10-100 μM.

[0016] Furthermore, when used as an antithrombotic drug, the final concentration of Veronica persica quinone A in the system is 0.35-1.4 mg / kg.

[0017] This invention proposes the use of the Veronica persicae in the preparation of medicaments for the treatment and / or prevention of thrombosis by inhibiting thrombosis with quinone A.

[0018] The application is characterized in that the Veronica persica quinone A is formulated with pharmaceutically acceptable excipients.

[0019] The application is characterized in that the dosage form of Veronica persica quinone A is an oral dosage form, a transdermal dosage form, an inhaler, or an injection.

[0020] The application is characterized in that the oral dosage form is a tablet, capsule, pill, or emulsion.

[0021] The HSP47 / PDI interaction small molecule inhibitor of the present invention has antithrombotic activity, which can fill the current gap in the application of HSP47 / PDI interaction small molecule inhibitors in antiplatelet aggregation and antithrombosis.

[0022] The present invention has the following advantages and beneficial effects:

[0023] The present invention relates to Veronica persica quinone A, which is the first to be discovered as an HSP47 / PDI interaction inhibitor. It can significantly inhibit thrombin-induced platelet aggregation in mice, has slight inhibitory activity on ADP-collagen-induced platelet aggregation, and has anti-platelet aggregation activity. Moreover, the inhibitory effect on thrombin-induced platelet aggregation is concentration-dependent.

[0024] According to existing technology, antithrombotic drugs mainly include anticoagulants, antiplatelet drugs, and fibrinolytic drugs, which are the main drugs for the prevention and treatment of thrombotic diseases. For example, aspirin has adverse reactions such as bleeding, including subcutaneous bleeding, gastrointestinal bleeding, and intracranial hemorrhage, which can lead to death or disability. Therefore, for patients with stroke, antithrombotic drugs cannot be taken long-term, as this may worsen their condition. Thus, finding drugs that do not cause bleeding while preventing thrombosis has always been a technical challenge in this field. This invention discovers that *Veronica persica* has antithrombotic activity against quinone A, effectively inhibiting FeCl3-induced carotid artery thrombosis in mice, while without a significant risk of bleeding.

[0025] Therefore, this invention provides a new approach and research foundation for the development of novel drugs for platelet activation and / or prevention and treatment of thrombotic diseases. Attached Figure Description

[0026] Figure 1 The results of the dose-dependent blocking effect of Veronica persica on the interaction between HSP47 and PDI proteins and the half-inhibition concentration determination of quinone A by Veronica persica in Example 1 are as follows:

[0027] Figure 2 This is a schematic diagram showing the results of Veronica persica in Example 2, which inhibited thrombin (A), collagen (B), and ADP (C)-induced platelet aggregation in mice.

[0028] Figure 3 This is a graph showing the effect of Veronica persicae on quinone A on platelet adhesion in Example 3; AD represents the results of fluorescence microscopy detection; E is a statistical graph of the expansion area of ​​platelets on collagen;

[0029] Figure 4 In Example 4, Veronica persica showed that quinone A could inhibit FeCl3-induced carotid artery thrombosis in mice. Figure 1 shows the observation graph, and Figure B shows the statistical graph of vascular occlusion time.

[0030] Figure 5 In Example 5, Veronica persica dose-dependently prolonged the tail bleeding time in mice in response to quinone A. Figure A shows the statistical results of tail bleeding time, Figure B shows the statistical results of bleeding absorbance, and Figure C shows the bleeding observation results. Detailed Implementation

[0031] The paraquinone A from Veronica persica was purchased from Chengdu Pusi Biotechnology Co., Ltd., batch number PS013805.

[0032] Example 1: The compound of the present invention inhibits the interaction between platelet heat shock protein 47 protein and protein disulfide isomerase.

[0033] Experimental Methods: Competitive Binding Inhibition Assay. 100 μL of 0.28 μg / mL HSP47 recombinant protein dissolved in coating buffer (50 mM carbonate buffer, pH 9.6) was added to a 96-well microplate and incubated overnight at 4°C. The next day, the plate was washed three times with 300 μL of washing buffer (phosphate buffer containing 0.05% Tween 20, pH 7.4) to remove uncoated protein. Then, 300 μL of blocking buffer (wash buffer containing 2% bovine serum albumin, pH 7.4) was added and the plate was blocked at 37°C for 1 h. After three washes, 50 μL of different concentrations of the test compound was mixed with 50 μL of 0.12 μg / mL biotinylated PDI recombinant protein and added to the 96-well plate, and incubated at 37°C for 1 h. After three washes, 100 μL of streptavidin-labeled horseradish peroxidase was added to each well and the plate was incubated at 37°C for 1 h. After rinsing three times, 100 μL of high-sensitivity chromogenic reagent was added to each well, and the mixture was incubated at 37°C in the dark for 20 minutes. After adding 50 μL of stop solution (2M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. The raw data were analyzed using GraphPadPrism 6.0 software, and the relative inhibition rate was calculated. The measured values ​​are expressed as the mean ± standard error of three independent experiments.

[0034] Experimental results: such as Figure 1 As shown, Veronica persica dose-dependently blocks the interaction between HSP47 and PDI recombinant proteins with quinone A, with a half-maximal inhibitory concentration (IC50) of 0.1665 μM. The compound Veronica persica of this invention, quinone A, can effectively inhibit the interaction between HSP47 and PDI proteins.

[0035] Example 2: Effects of Veronica persica on quinone A on thrombin, collagen, and ADP-induced platelet aggregation in mice.

[0036] Experimental Methods: Blood was drawn from the abdominal aorta of rats and anticoagulated with sodium citrate at a ratio of 1:9. The blood was centrifuged at 800 rpm for 15 min at room temperature, and the supernatant platelet-rich plasma (PRP) was collected. This was repeated twice, and the two PRP samples were combined. The remaining plasma was centrifuged at 3000 rpm for 10 min at room temperature, and the supernatant platelet-poor plasma (PPP) was collected. The combined PRP was then centrifuged again at 800 rpm for 10 min at room temperature to remove as many residual white blood cells and red blood cells as possible. The platelet count in the PRP was counted using a platelet counter, and the platelet count was adjusted to approximately 2 × 10⁻⁶ using PPP. 8 / mL.

[0037] Thirty minutes before the experiment, the platelet aggregator was turned on and preheated to 37°C. First, the optical path of several channels was checked using double-distilled water. 320 μl of PRP was added to the aggregation tubes, followed by the addition of Veronica persica-p-quinone A at final concentrations of 10-100 μM. The tubes were incubated at 37°C with stirring at 600 rpm for 5 minutes. The instrument was zeroed using PPP to adjust the baseline. Once the baseline stabilized, agonists such as collagen (final concentration 17 μg / mL), thrombin (final concentration 25 U / mL), or ADP (final concentration 25 μM) were added, and platelet aggregation was detected. The degree of platelet aggregation was recorded by the platelet aggregator over 5-10 minutes, and the maximum aggregation degree (MA) during the aggregation process was also recorded.

[0038] Experimental results: such as Figure 2 As shown, 10-100 μM Veronica persica quinone A inhibited thrombin, collagen and ADP-induced platelet aggregation in rats compared with the control group, and the inhibitory effect on thrombin-induced platelet aggregation was concentration-dependent.

[0039] Example 3: The process by which Veronica persica inhibits platelet adhesion by quinone A

[0040] Experimental Method: Cover slips were placed in 24-well plates and coated overnight at 4°C with 100 μL of 5 μg / mL collagen. The coating solution was aspirated, and the plates were washed three times with pre-cooled PBS. After blocking with 1% (w / v) BSA for 2 h, the plates were washed again with PBS. Platelets (2 × 10⁻⁶) pre-incubated for 10 min with control solvent or different concentrations of drug were added. 7 Add CaCl2 (final concentration 1 mM) to the sample (particles / mL) and incubate at 37°C for 1 h. Then aspirate the residual liquid and wash with pre-warmed PBS (37°C). Fix with pre-warmed PBS containing 4% (w / v) paraformaldehyde at room temperature for 10 min, then permeate with PBS containing 0.1% (w / v) Triton X-100 for 5 min. Wash three times with PBS, then add FITC-labeled phalloidin (final concentration 200 nM) and incubate in the dark for 30 min. After washing with PBS, mount with anti-fluorescence quenching mounting medium. Observe under a fluorescence microscope, randomly photograph 8 fields, and quantify using ImageJ software. Analyze the raw data using GraphPad Prism 6.0 software. One-way ANOVA was used for intergroup analysis, with p < 0.05 considered statistically significant, p < 0.01 considered highly statistically significant, and p < 0.001 considered extremely statistically significant.

[0041] Experimental results: During thrombosis, platelet aggregation can be divided into three stages: platelet adhesion, expansion, and stabilization. Platelet adhesion refers to the process where, after endothelial cell damage, circulating platelets adhere to the wound site through the binding of GPVI-IX-V and VWF-collagen complex.

[0042] Figure 3 The results showed that, compared with the control group, platelet activation was significantly inhibited and the adhesion area to collagen was significantly reduced after treatment with Veronica persica quinone A.

[0043] Example 4: Veronica persica inhibits FeCl3-induced carotid artery thrombosis in mice by inhibiting quinone A.

[0044] Experimental Methods: FeCl3-induced carotid artery thrombosis model in mice. The test animals were male ICR mice, housed separately in clean-grade animal laboratories at the Animal Center of China Pharmaceutical University before the experiment. The mice were divided into five groups: a model group, an aspirin group, and high, medium, and low doses of the compound described in this invention, with six mice in each group. The dosages were as follows: model group received physiological saline containing 1% DMSO and 1% Tween 80; aspirin group received 20 mg / kg; and the high, medium, and low doses of the compound were 0.5 mg / kg, 1 mg / kg, and 2 mg / kg, respectively. The compound was dissolved in DMSO and Tween 80 and diluted with physiological saline to prepare a solution containing 1% DMSO and 1% Tween 80. Administration was administered intravenously, with model initiation occurring 1 hour after intravenous injection. The modeling method involved anesthetizing mice with 5% chloral hydrate. The skin of the mouse neck was longitudinally incised along the midline using sterile instruments. Surgical forceps were used to separate the muscle tissue, exposing the trachea. The lateral carotid artery was isolated approximately 3 cm. A plastic film was used to separate the carotid artery from the surrounding tissue to protect the surrounding blood vessels. A small filter paper soaked in 7.5% FeCl3 solution was placed tightly against the carotid artery for 2 minutes. The blood vessels were then imaged using a moorFLPI-2 laser speckle blood flow imaging system. One vascular image was captured every minute. Each mouse was sacrificed after 30 minutes of imaging. Blood flow per minute was analyzed using mFLPI2 software, and the time to loss of blood flow was recorded. The raw data were analyzed using GraphPadPrism 6.0 software. Measured values ​​are expressed as the mean ± standard error of the mean for each independent experiment. One-way ANOVA was used for inter-group analysis, and p < 0.05 was considered statistically significant.

[0045] Experimental results: The compound can inhibit FeCl3-induced carotid artery thrombosis in mice. Figure 4As shown in Figure A, which illustrates the observation of FeCl3-induced carotid artery thrombosis in mice, and Figure B, which shows the average time to disappearance of blood flow in the model group (6 min), the average time to disappearance of blood flow in the positive control drug aspirin group (16 min), and the average times to disappearance of blood flow in the high, medium, and low dose groups of the compound (8 min, 11 min, and 25 min, respectively). Compared with the model group, the positive control drug and the compound significantly prolonged the time to disappearance of blood flow, indicating a clear dose-dependent effect.

[0046] Example 5: Veronica persica showed no significant bleeding risk to quinone A.

[0047] Experimental Methods: Tail docking experiment. The test animals were male ICR rats, housed separately in clean-grade animal laboratories at the Animal Center of China Pharmaceutical University before the experiment. The mice were divided into five groups: a model group, an aspirin group, and high, medium, and low doses of the compound described in this invention, with six mice in each group. The dosages were as follows: model group received physiological saline containing 1% DMSO and 1% Tween 80; aspirin group received 20 mg / kg; and the high, medium, and low doses of the compound were 0.35 mg / kg, 0.7 mg / kg, and 1.4 mg / kg, respectively. The compound was dissolved in DMSO and Tween 80 and diluted with physiological saline to prepare a solution containing 1% DMSO and 1% Tween 80. Administration was administered intravenously. One hour after administration, the mice were placed in a tail vein injection device. A culture dish containing 4 ml of physiological saline was prepared, and the tail was docked 2 mm from the tip. The docked tail was placed in the culture dish, and the bleeding time was recorded from the onset of blood flow until it stopped. Physiological saline was collected after bleeding, cells were sonicated and centrifuged at 10,000 rpm for 5 min, the supernatant was collected, and absorbance was measured to indicate the amount of bleeding. Raw data were analyzed using GraphPadPrism 6.0 software. Measured values ​​are expressed as the mean ± standard error of the mean for each independent experiment. One-way ANOVA was used for between-group analysis, and p < 0.05 was considered statistically significant.

[0048] Experimental results: Low and medium doses of the compound described in this invention did not pose a significant risk of bleeding. For example... Figure 5 As shown, the average tail bleeding time in the model group mice was 120 s, in the aspirin group it was 464 s, and in the low, medium, and high dose groups of the compound described in this invention the average bleeding times were 148 s, 271 s, and 652 s, respectively. Compared with the low and medium doses in the model group, aspirin had a significant bleeding risk, while the bleeding time in the low and medium dose groups of the compound described in this invention showed little difference from that in the model group, indicating that the low and medium doses of the compound had virtually no effect on the coagulation function of mice and no significant bleeding risk.

Claims

1. The use of Veronica perforata quinone A in the preparation of drugs for the prevention or treatment of thrombosis, wherein the chemical structural formula of Veronica perforata quinone A is shown below:

2. The application according to claim 1, characterized in that, The drug is a formulation of Veronica perforata quinone A and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The drug dosage form is an oral preparation, a transdermal preparation, an inhaler, or an injection.

4. The application according to claim 3, characterized in that, The oral administration formulation is a tablet, capsule, pill, or emulsion.

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