Application of procumbent speedwell p-quinone A in preparation of medicine for preventing or treating thromboembolic diseases

Abstract

The invention relates to a novel application of veronica lanceolata p-quinone A, in particular to an application of veronica lanceolata p-quinone A in preparation of a medicine for preventing or treating thromboembolic diseases. The invention finds that the procumbent speedwell p-quinone A can remarkably inhibit platelet aggregation activity induced by different in-vitro inducers; in an in-vivo thrombus model experiment, the procumbent speedwell p-quinone A can well inhibit mouse carotid artery thrombosis induced by FeCl3. Meanwhile, the prosperous speedwell p-quinone A disclosed by the invention does not have an obvious bleeding tendency. The prosperous speedwell p-quinone A is expected to play a certain positive role in prevention or treatment of thromboembolic diseases.

Description

Technical Field

[0001] The invention relates to a new application of Veronica verticillata quinone A, and in particular to an application of Veronica verticillata quinone A in preparing a medicine for preventing or treating thromboembolic diseases. Background Art

[0002] Thromboembolic diseases seriously affect human health worldwide and have become a major health problem of worldwide concern. The development process includes two stages: thrombosis and thromboembolism. The types of thrombosis in the initial stage can be divided into four types: white thrombus (i.e. platelet thrombus), mixed thrombus, red thrombus, and transparent thrombus. Platelet thrombus is the first step in thrombosis, so antiplatelet therapy is the cornerstone of the treatment of thromboembolic diseases. The formation of platelet thrombus is a multi-step process. Under normal circumstances, platelets do not interact with intact vascular walls. However, in tissue trauma, platelets adhere to the extracellular matrix in the process of coordinated action of ligands of platelet surface receptors such as von Willebrand factor (vWF), collagen, laminin, glycoprotein Ibα (GP Ibα), and thrombospondin, resulting in the initial tethering and rolling of platelets on the damaged vascular wall. The final step of platelet adhesion requires the integrin αIIbβ3 to change from a low-affinity state to a high-affinity state, mediating the final firm adhesion of platelets by binding to vWF and fibrinogen. Platelet adhesion triggers a signaling cascade mediated by tyrosine kinases and G protein-coupled receptors, which guides the full activation of platelets and the accompanying release of granules, leading to the recruitment and activation of other platelets. Platelet adhesion and activation lead to platelet aggregation and the appearance of a procoagulant surface, promoting the formation of a fibrin-rich hemostatic plug at the site of injury. Currently, commonly used antiplatelet drugs in clinical practice, such as aspirin, clopidogrel, and ticagrelor, can directly and irreversibly inhibit platelet activation and aggregation by inhibiting platelet cyclooxygenase (COX)-1, P2Y12 receptors, etc., and have a significant therapeutic effect in inhibiting thrombosis, but at the same time have a higher risk of bleeding. Therefore, it is crucial to find a way to inhibit thrombosis without affecting bleeding.

[0003] Protein-protein interactions (PPIs) are the basic units of life activities and play a core role in the physiological and pathological processes of all organisms. Dysregulation of PPI function is the basis of many diseases, including thrombosis, cancer, immune diseases, etc., so PPI is a very direct and effective drug target. In the application of platelet diseases, since PPI does not directly inhibit platelet activation receptors or coagulation factors, the search for traditional Chinese medicine small molecule inhibitors that act on platelet PPI has lower bleeding risk and safety.

[0004] Protein disulfide isomerase (PDI) plays an important role in the process of thrombosis. PDI binds to activated platelets, thereby mediating the conformational transition of integrin αIIbβ3, causing integrin αΙΙbβ3 to change from a low-affinity to a high-affinity state, enhancing its binding ability with fibrinogen and promoting thrombosis. Integrin αΙΙbβ3 plays a vital role in the signal transduction of platelet activation and aggregation. Direct inhibition of the activity of integrin αΙΙbβ3 will lead to serious bleeding risks. Therefore, targeting extracellular PDI may be a therapeutic strategy for the treatment of thrombotic diseases. In theory, since PDI does not directly block platelet activation receptors or coagulation factors, it has a low bleeding risk and high safety. Researchers have conducted many studies on the discovery of PDI inhibitors and the development of PDI monoclonal antibodies. Most of the PDI inhibitors discovered so far are irreversible inhibitors, such as CxxC peptides, bacillin, P1 and TA. In the regulation of thrombotic diseases, reversible inhibitors help maintain the dynamic balance of the body's anticoagulation and coagulation functions, so reversible PDI inhibitors are a better choice. The reversible PDI inhibitors with antithrombotic effects reported in the literature are mainly flavonoids, such as rutin, quercetin, myricetin, etc. Among them, rutin is the best PDI inhibitor screened by high-throughput screening in the small molecule compound library. Intravenous injection of rutin can reduce the volume of arterial thrombi caused by laser damage but does not increase bleeding time. However, some studies have shown that rutin is a non-specific inhibitor. In the process of using PDI monoclonal antibody treatment, Jaegyung et al. found that PDI monoclonal antibodies prolonged the bleeding time of the mouse tail and increased blood loss, which raised concerns that selective PDI inhibitors may interfere with hemostasis. In addition, cell-permeable molecular inhibitors may cause serious side effects because PDI plays 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 has a low binding rate to non-activated platelets, and its binding degree increases with platelet activation. It is still unclear how PDI activity is regulated during thrombosis, so we tried to find molecules that interact with PDI and may regulate PDI activity during platelet activation.

[0005] It has been reported in the literature that HSP47 is expressed on the surface of human platelets. After GPVI activation, HSP47 expression increases by 48% and enhances the interaction between platelets and collagen. A recently published literature study confirmed that downregulating heat shock protein 47 (HSP47) on the surface of platelets has an anti-thrombotic effect. The article found a variety of differential proteins in the platelets of hibernating brown bears based on mass spectrometry proteomics, among which HSP47 was downregulated 55 times. After HSP47 was downregulated, the activation of platelet activation-related receptors such as integrin αIIbβ3 and glycoprotein VI was significantly downregulated. In addition, downregulation of HSP47 weakened the activation of immune cells and the formation of neutrophil extracellular traps, inhibiting thrombosis in hibernating brown bears, mice and patients with spinal cord injury. Literature studies have shown that HSP47 released after platelet activation binds to the platelet surface, enhances the binding function of platelet surface molecular ligands, and promotes thrombosis, but its mechanism of action has not been reported in the literature. Through the String protein interaction database search and molecular docking, the results show that there may be a direct interaction between HSP47 and PDI. The results of the immunoprecipitation experiment show that HSP47 can form a molecular complex with PDI. The results of the platelet aggregation experiment show that increasing the concentration of HSP47 and PDI in vitro can promote platelet aggregation. The above experimental results prove that platelet HSP47 interacts with PDI and promotes platelet aggregation. Since the traditional Chinese medicine small molecule inhibitors acting on platelet PPI have lower bleeding risk and safety, the present invention focuses on finding small molecules that inhibit the interaction between platelet HSP47 and PDI.

[0006] The traditional Chinese medicine Danshen is the dried root or rhizome of Salvia miltiorrhiza Bge., a plant of the Labiatae family. It is slightly cold in nature, bitter in taste, and enters the heart meridian and liver meridian. It is an important medicine for promoting blood circulation and removing blood stasis in traditional Chinese medicine. Danshen contains lipophilic terpenes and water-soluble phenolic acids, fatty acids, lactones, polysaccharides, flavonoids, quinones and other components. Sibiriquinone A is a quinone compound obtained from Danshen after ligand affinity fishing with protein HSP47 and protein PDI. Literature studies have shown that Sibiriquinone A has a strong inhibitory effect on lymphocyte transformation, and other pharmacological studies have rarely been reported, especially the effect of Sibiriquinone A on platelet function and thrombotic diseases has not been reported. Summary of the invention

[0007] Purpose of the invention: One of the purposes of the present invention is to provide a new use of Veronica rotundifolia quinone A in the preparation of anti-platelet aggregation and anti-thrombosis drugs, so as to expand the application scope of Veronica rotundifolia quinone A.

[0008] Technical solution:

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

[0010]

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

[0012] Application of Veronica verticillata quinone A in preparing medicine for treating or preventing thrombosis.

[0013] Application of Veronica verticillata quinone A in preparing drugs for treating or preventing platelet aggregation.

[0014] Application of Veronica verticillata quinone A in preparing medicine for preventing or treating thromboembolic diseases.

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

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

[0017] The present invention proposes the use of the Veronica verticillata quinone A in inhibiting thrombosis in the preparation of a medicine for treating and / or preventing thrombosis.

[0018] The application is characterized in that the Veronica quinone A is combined with pharmaceutically acceptable excipients to form a preparation.

[0019] The application is characterized in that the dosage form of Veronica verticillata quinone A is an oral dosage form, a transdermal dosage form, an inhalation, 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 anti-thrombotic activity, and can fill the gap that there is no HSP47 / PDI interaction small molecule inhibitor currently used in anti-platelet aggregation and anti-thrombosis.

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

[0023] The Veronica quinone A involved in the present invention is firstly found to be an HSP47 / PDI interaction inhibitor, which can significantly inhibit thrombin-induced mouse platelet aggregation, has a slight inhibitory activity on ADP collagen-induced platelet aggregation, and has anti-platelet aggregation activity, and the inhibitory effect on thrombin-induced platelet aggregation is concentration-dependent.

[0024] According to the prior art, antithrombotic drugs mainly include anticoagulants, antiplatelet drugs and fibrinolytic drugs, which are the main drugs for preventing and treating thrombotic diseases, such as aspirin, whose adverse reactions are bleeding phenomena, such as subcutaneous bleeding, gastrointestinal bleeding, intracranial bleeding, which can cause death or disability, etc. Therefore, for patients with cerebral infarction, antithrombotic drugs cannot be taken for a long time, which may cause the condition to worsen. Therefore, it has always been a technical problem to be solved in this field to find drugs that do not cause bleeding phenomena and resist thrombosis. The present invention finds that Veronica verticillata has antithrombotic activity against quinone A, which can effectively inhibit FeCl3-induced carotid artery thrombosis in mice, and there is no obvious risk of bleeding.

[0025] Therefore, the present invention provides a new idea and research basis for the development of new drugs for platelet activation and / or prevention and treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the determination of the half-inhibitory concentration of Veronica verticillata on the dose-dependent blocking effect of quinone A on the interaction between HSP47 and PDI protein in Example 1;

[0027] Figure 2 This is a schematic diagram of the results of the inhibition of rat platelet aggregation induced by thrombin (A), collagen (B), and ADP (C) by Veronica verticillata quinone A in Example 2;

[0028] Figure 3 is the result diagram of the effect of Veronica verticillata quinone A on platelet adhesion in Example 3; AD is the result of fluorescence microscope detection; E is the statistical diagram of the expansion area of ​​platelets on collagen;

[0029] Figure 4 The Veronica quinone A in Example 4 can inhibit FeCl3-induced carotid artery thrombosis in mice. Figure A is an observation graph, and Figure B is a statistical graph of vascular occlusion time;

[0030] Figure 5 The Veronica verticillata quinone A in Example 5 can prolong the tail bleeding time of mice in a dose-dependent manner. Figure A is the statistical result of tail bleeding time, Figure B is the statistical result of bleeding absorbance, and Figure C is the bleeding observation result. DETAILED DESCRIPTION

[0031] Veronica quinone A was purchased from Chengdu Pusi Biotechnology Co., Ltd. with batch number PS013805.

[0032] Example 1 The compounds of the present invention inhibit the interaction between platelet heat shock protein 47 protein and protein disulfide isomerase

[0033] Experimental method: Competitive binding inhibition experiment. 100 μL of 0.28 μg / mL HSP47 recombinant protein dissolved in coating solution (50 mM carbonate buffer, pH 9.6) was added to a 96-well microplate and placed overnight at 4°C. The next day, the plate was rinsed three times with 300 μL of washing solution (phosphate buffer containing 0.05% Tween20, pH 7.4) to remove uncoated proteins. Subsequently, 300 μL of blocking solution (washing solution containing 2% bovine serum albumin, pH 7.4) was added and blocked at 37°C for 1 hour. After rinsing three times, 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 hour. After rinsing three times, 100 μL of streptavidin-labeled horseradish peroxidase was added to each well and incubated at 37°C for 1 hour. After rinsing three times, 100 μL of high-sensitivity colorimetric solution was added to each well and incubated at 37°C in the dark for 20 minutes. After adding 50 μL of reaction stop solution (2M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. The raw data was analyzed using GraphPadPrism 6.0 software, and the relative inhibition rate was calculated. The measured values ​​are expressed as the mean ± standard error of the mean of three independent experiments.

[0034] Experimental results: Figure 1 As shown, Veronica quinone A dose-dependently blocked the interaction between HSP47 and PDI recombinant protein, with a half inhibition concentration of 0.1665 μM. The compound Veronica quinone A of the present invention can effectively inhibit the interaction between HSP47 protein and PDI protein.

[0035] Example 2 Effect of Veronica rotundifolia Quinone A on Thrombin, Collagen and ADP-Induced Rat Platelet Aggregation

[0036] Experimental method: Blood was drawn from the abdominal aorta of rats, and sodium citrate was used as an anticoagulant at a ratio of 1:9. The upper layer of platelet-rich plasma (PRP) was collected by centrifugation at 800 rpm at room temperature for 15 minutes, and the upper layer of platelet-rich plasma (PRP) was collected by centrifugation at 800 rpm at room temperature for 15 minutes again. The two layers of platelet-rich plasma were combined, and the remaining plasma was centrifuged at 3000 rpm at room temperature for 10 minutes to collect the upper layer of platelet-poor plasma (PPP). The combined PRP was centrifuged again at 800 rpm at room temperature for 10 minutes to remove residual white blood cells and red blood cells as much as possible. The number of platelets in PRP was counted using a platelet counter, and the number of platelets in PRP was adjusted to approximately 2×10 using PPP. 8 / mL.

[0037] Turn on the platelet aggregation instrument 30 minutes before the experiment, preheat the platelet aggregation instrument to 37°C, and then use double distilled water to detect the optical path of several channels. Take 320μl PRP and add it to the aggregation tube, add the final concentration of 10-100μM of Veronica quinone A, and incubate at 37°C with a stirring speed of 600rpm for 5min. Use PPP to zero the instrument and adjust the baseline. After the baseline remains stable, add the agonist with a final concentration of 17μg / mL collagen or 25U / mL thrombin or 25μM ADP to detect platelet aggregation. The degree of platelet aggregation is recorded by the platelet aggregation instrument for 5-10min, and the maximum aggregation degree (MA) of platelets during the aggregation process is recorded.

[0038] Experimental results: Figure 2 As shown, 10-100 μM of Veronica quinone A in rat platelets can inhibit thrombin, collagen and ADP-induced platelet aggregation compared with the control group, and the inhibitory effect on thrombin-induced platelet aggregation is concentration-dependent.

[0039] Example 3 Veronica rotundifolia inhibits platelet adhesion by quinone A

[0040] Experimental method: Coverslips were placed in 24-well plates and coated with 100 μL of 5 μg / mL collagen at 4°C overnight. The coating solution was removed and 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 7 / mL), then add CaCl2 (final concentration of 1mM), and incubate in a 37℃ incubator for 1h. Then remove the residual liquid and wash with PBS preheated at 37℃. Fix with preheated PBS containing 4% (w / v) paraformaldehyde at room temperature for 10min, permeate with PBS solution containing 0.1% (w / v) Triton X-100 for 5min, wash three times with PBS, add FITC-labeled phalloidin (final concentration of 200nM) and incubate in the dark for 30min. After washing with PBS, seal the slide with anti-fluorescence quenching sealing liquid. Observe under a fluorescence microscope, randomly shoot 8 fields of view, and quantify with Image J software. Use GraphPad Prism 6.0 software to analyze the raw data. One-way analysis of variance was used for inter-group analysis, with p<0.05 as statistically significant, p<0.01 as significant statistically significant, and p<0.001 as extremely significant statistically significant.

[0041] Experimental results: During the thrombosis process, platelet aggregation can be divided into three stages: platelet adhesion, expansion and stabilization. The platelet adhesion process refers to the adhesion of circulating platelets to the wound site through the combination of GPVI-IX-V and VWF-collagen complex after endothelial cells are damaged.

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

[0043] Example 4 Veronica vertebrata quinone A inhibits FeCl3-induced carotid artery thrombosis in mice

[0044] Experimental method: FeCl3-induced mouse carotid artery thrombosis model experiment. The test animals were ICR male mice, which were caged and kept in the clean animal experiment of the Animal Center of China Pharmaceutical University before the experiment. The test mice were divided into 5 groups, including the model group, the aspirin group, and the high, medium and low doses of the compound of the present invention, with 6 mice in each group. The dosage was respectively 1% DMSO and 1% Tween 80 in the model group, 20 mg / kg in the aspirin group, and 0.5 mg / kg, 1 mg / kg and 2 mg / kg in the high, medium and low doses of the compound, respectively. The compounds used were dissolved in DMSO and Tween 80, and diluted with saline to prepare a solution containing 1% DMSO and 1% Tween 80. The administration time was in advance, the administration method was intravenous injection, and the model was established 1 hour after intravenous injection. The modeling method is to anesthetize the test mice with 5% chloral hydrate, cut the mouse neck skin longitudinally along the midline of the neck with sterilized equipment, separate the muscle tissue with surgical curved forceps, expose the trachea, separate the lateral carotid artery about 3cm, separate the carotid artery from the surrounding tissue with a plastic film to protect the surrounding vascular tissue, and put a small filter paper with 7.5% FeCl3 solution close to the carotid artery. After applying the paper for 2 minutes, the blood vessels were imaged under the moorFLPI-2 laser speckle blood flow imager. One vascular imaging image was taken every minute, and each mouse was killed after 30 minutes of shooting. The blood flow per minute was analyzed using mFLPI2 software, and the time of blood flow disappearance was counted. The original data were analyzed using GraphPadPrism6.0 software. The measured values ​​are expressed as the mean ± standard error of the mean for each independent experiment. One-way analysis of variance 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, Figure A shows the observation diagram of FeCl3-induced carotid artery thrombosis in mice, and Figure B shows that the average time of blood flow disappearance in the model group is 6 minutes, the average time of blood flow disappearance in the positive drug aspirin group is 16 minutes, and the average time of blood flow disappearance in the high, medium and low dose groups of the compound is 8 minutes, 11 minutes, and 25 minutes, respectively. Compared with the model group, the positive drug and the compound can significantly prolong the time of blood flow disappearance, and the compound has obvious dose dependence.

[0046] Example 5: Veronica rotundifolia quinone A has no significant bleeding risk.

[0047] Experimental method: tail-cut experiment. The test animals were ICR male mice, which were caged and kept in the clean animal experiment of the Animal Center of China Pharmaceutical University before the experiment. The test mice were divided into 5 groups, including model group, aspirin group, high, medium and low doses of the compound of the present invention, with 6 mice in each group. The dosage was respectively 1% DMSO and 1% Tween 80 in the model group, 20 mg / kg in the aspirin group, 0.35 mg / kg, 0.7 mg / kg and 1.4 mg / kg in the high, medium and low doses of the compound, respectively. The compounds used were dissolved in DMSO and Tween 80, and the saline was diluted to form a solution containing 1% DMSO and 1% Tween 80. The administration time was in advance, and the administration method was intravenous injection. The mouse was placed in the tail vein injection instrument. One hour after the administration, a culture dish containing 4 ml of saline was prepared, and the tail tip of the mouse was cut off at 2 mm. The cut tail was placed in the culture dish, and the timing started from the outflow of blood until the blood flow stopped, and the bleeding time was counted. After bleeding, physiological saline was collected, cells were ultrasonically disrupted, centrifuged at 10,000 rpm for 5 min, the supernatant was taken, and the absorbance was measured to indicate the amount of bleeding. The raw data were analyzed using GraphPad Prism 6.0 software. The measured values ​​were expressed as the mean ± standard error of the mean for each independent experiment. One-way analysis of variance was used for inter-group analysis, and p < 0.05 was considered statistically significant.

[0048] Experimental results: The compounds of the present invention have no significant bleeding risk at medium and low doses. Figure 5 As shown, the average bleeding time of the tail of mice in the model group was 120s, that in the aspirin group was 464s, and the average bleeding time in the low, medium and high dose groups of the compound of the present invention were 148s, 271s and 652s, respectively. Compared with the low and medium doses in the model group, aspirin has an obvious bleeding risk, while the bleeding time in the low and medium dose groups of the compound is less significantly different from that in the model group, indicating that the low and medium dose compounds have little effect on the coagulation function of mice and no obvious bleeding risk.

Claims

1. Use of Veronica quinone A in the preparation of an inhibitor of the interaction between heat shock protein 47 and protein disulfide isomerase, wherein the chemical structural formula of Veronica quinone A is as follows: Veronica quinone A.

2. Application of Veronica quinone A in the preparation of drugs for preventing or treating thrombosis.

3. Application of Veronica rotundifolia quinone A in the preparation of drugs for preventing or treating platelet aggregation.

4. Application of Veronica quinone A in the preparation of drugs for preventing or treating thromboembolic diseases.

5. The use according to any one of claims 1 to 3, characterized in that: The Veronica quinone A is a preparation of Veronica quinone A and pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The dosage form of Veronica verticillata quinone A is oral preparation, transdermal preparation, inhalation or injection.

7. The use according to claim 6, characterized in that: The oral administration preparation is a tablet, capsule, pill or emulsion.

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