Application of ester compound in preparation of antithrombotic drugs

Abstract

The invention discloses an application of an ester compound in preparation of an antithrombotic drug. The structural formula of the ester compound is as shown in the following formula: # imgabs0 #. The ester compound can reduce blood clot formation in thrombus tissues by inhibiting TF expression and improve thrombus-related pathological processes by regulating the level of inflammatory factors (such as TXB2 and 6-keto-PGF1 alpha), and is used for preparing antithrombotic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of an ester compound in the preparation of an anti-thrombotic drug. Background Art

[0002] Thrombotic diseases are the result of the imbalance of the processes of promoting coagulation, anticoagulation, and fibrinolysis. Currently, the clinical drugs used for the prevention and treatment of thrombotic diseases include anticoagulants, antiplatelet drugs, and thrombolytic drugs. Among them, anticoagulants account for the majority. However, due to the extremely high bleeding risk and short treatment time window of anticoagulants, the use of anticoagulants is greatly restricted. Therefore, actively exploring safe and effective drugs for preventing and treating thrombosis is a hot and difficult point in the research of thrombotic diseases.

[0003] Tissue factor (TF) is a single-chain transmembrane glycoprotein with a molecular weight of 47 kD, and it is the only coagulation factor present in cells and tissues outside normal human plasma. When a blood vessel is damaged, the blood is exposed to the cell surface that can express TF. Subsequently, TF forms a complex with its ligand FⅦ or FⅦa, which further activates coagulation factor IX and coagulation factor X in the blood, and simultaneously initiates the extrinsic and intrinsic coagulation pathways, further activating prothrombin (FII) and fibrinogen, and then forming a blood clot with activated platelets. Therefore, TF is the most important initiating factor in the coagulation process. Under the stimulation of pathological factors, vascular endothelial cells and monocytes in the blood are induced to express TF, thus participating in the formation of various pathological thrombi. Therefore, TF is not only an important initiating factor for physiological hemostasis but also a key factor for pathological thrombus formation. By regulating TF, thrombus formation can be effectively inhibited. Summary of the Invention

[0004] One object of the present invention is to provide the application of the compound shown in the following formula in the preparation of an anti-thrombotic drug,

[0005]

[0006] Further, the drug reduces the expression of tissue factor.

[0007] Further, the drug reduces the formation of blood clots.

[0008] Further, the drug regulates the expression of inflammatory factors.

[0009] Another object of the present invention is to provide the application of a pharmaceutical composition in the preparation of an anti-thrombotic drug. The pharmaceutical composition includes an active ingredient, and the active ingredient is the compound shown in the following formula:

[0010]

[0011] In the present invention, the administration route applied can be oral administration or injection, specifically intraperitoneal, subcutaneous, intravenous or intramuscular injection; pharmaceutically acceptable carriers such as diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. can also be added to prepare a pharmaceutical composition in a certain form, such as powders, pills, capsules, tablets, microcapsules, soft capsules, films, suppositories, injections, ointments, powders, granules, aerosols and other preparations.

[0012] The present invention proves through experiments that this compound has a significant antithrombotic effect in a mouse carotid artery thrombosis model induced by ferric chloride (FeCl 3 ). This compound can inhibit TF expression, reduce the formation of blood clots in thrombus tissues, and improve the pathological processes related to thrombosis by regulating the levels of inflammatory factors (such as TXB2, 6-keto-PGF1α). The present invention not only reveals the pharmacological effects of this compound in thrombotic diseases, but also provides an important basis for the development of safe and highly effective new therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effects of different doses of Compound No. 6 on ferric chloride-induced carotid artery thrombosis in mice. Among them, A is the diagram of carotid artery thrombus, B is the statistical chart of carotid artery thrombus weight, C is the H&E staining diagram of carotid artery thrombus tissue (n = 6, expressed as mean ± SEM, ### P < 0.001 compared with the sham operation group, ** P < 0.01 and *** P < 0.001 compared with the model group);

[0014] Figure 2 Effects of different doses of Compound No. 6 on the expression of tissue factor in carotid artery thrombus tissues of ferric chloride-induced mice. Among them, A is the immunohistochemical staining diagram of tissue factor in carotid artery thrombus tissues, B is the immunohistochemical statistical chart of tissue factor, C is the immunofluorescence staining diagram of tissue factor in carotid artery thrombus tissues, D is the immunofluorescence statistical chart of tissue factor (n = 6, expressed as mean ± SEM, ### P < 0.001 compared with the sham operation group, * P < 0.05 and *** P < 0.001 compared with the model group);

[0015] Figure 3 Effects of different doses of Compound No. 6 on the contents of coagulation factors and inflammatory factors in plasma of carotid artery thrombus mice induced by ferric chloride. Among them, A is the statistical chart of tissue factor (TF), B is thromboxane B2 (TXB 2)Statistical chart. C is the statistical chart of 6-keto-prostaglandin F1α (6-keto-PGF1α) (n = 6, expressed as mean ± SEM, ### P < 0.001 compared with the sham operation group, * P < 0.05, ** P < 0.01 and *** P < 0.01 compared with the model group). Detailed implementation manners

[0016] Currently, the therapeutic drugs for thrombotic diseases mainly focus on anticoagulant and thrombolytic drugs. However, these drugs have significant deficiencies in clinical applications. Anticoagulant drugs act by inhibiting the coagulation process. Although they can reduce thrombus formation, their high bleeding risk poses a major threat to the safety of patients, especially elderly patients or patients during postoperative recovery. In addition, thrombolytic drugs are mostly used for acute-phase treatment, with a short treatment window and limited efficacy, failing to meet the needs of patients with chronic thrombotic diseases. On the other hand, the existing drugs have limited effects on regulating the pathological mechanism of thrombosis. Currently, there is no specific targeted drug for TF available, and the overexpression of TF has been proven to be a key factor in thrombus formation. At the same time, the improvement effect of traditional drugs on thrombus-related inflammatory responses is also relatively limited, which further restricts the improvement of clinical efficacy. Therefore, developing therapeutic drugs for thrombotic diseases with clear action mechanisms and safety and effectiveness has become a medical problem that urgently needs to be solved. Through research, the present invention finds that the ester compound (Compound 6) can inhibit thrombus formation through multiple mechanisms, including reducing TF expression, decreasing the level of TXB2 in plasma, and increasing the content of 6-keto-PGF1α, thereby providing a new technical means for the prevention and treatment of thrombotic diseases.

[0017] The research of the present invention shows that the ester compound (Compound 6) effectively inhibits thrombus formation by intervening in the thrombus formation process, including inhibiting the expression of the key initiator TF, decreasing the content of the pro-thrombotic factor TXB2 in plasma, and significantly increasing the level of the anti-thrombotic factor 6-keto-PGF1α. In the mouse carotid artery thrombus model induced by ferric chloride (FeCl 3 )), Compound 6 shows significant anti-thrombotic effects. Especially at a high dose (3 mg / kg), its efficacy is better than that of the positive drug group, showing good dose-dependent characteristics.

[0018] Specific experimental results showed that Compound 6 could effectively reduce the volume and weight of thrombus formation, and its improvement effect on the pathological structure of thrombus tissue was confirmed by histological examination. In terms of thrombus-related inflammatory factors, Compound 6 reduced platelet activation by decreasing the TXB2 level, and enhanced vascular protection by increasing the 6-keto-PGF1α level. In addition, this compound could also improve the vascular tissue inflammatory response caused by thrombus and significantly reduce the pathological damage score.

[0019] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0022] Example 1

[0023] Effect of Different Doses of Compound 6 on Ferric Chloride-Induced Carotid Artery Thrombosis in Mice

[0024] I. Experimental Materials

[0025] 1. Instruments and Reagents

[0026] 5-0 and 6-0 medical silk braided threads were purchased from Pudong Jinhuan Medical Supplies Co., Ltd., sterilized medical suture needles were purchased from Chaohu Binxiong Medical Instrument Co., Ltd., tissue scissors, wire scissors, surgical microscissors, micro forceps, needle holders, and glass microspatulas were purchased from Shanghai Surgical Instrument Factory, filter paper was purchased from Whatman Company, AE240 electronic balance was purchased from METTLER Company in Switzerland, refrigerated centrifuge (Z-323K) was purchased from BECKMAN Company, ultra-low temperature refrigerator was purchased from Thermo Company in the United States, Milli-Q ultrapure water instrument was purchased from Millipore Company in the United States, constant temperature oven was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd., vortex mixer was purchased from Qilinbeier Instrument Co., Ltd. in Haimen, and Infinite200Pro microplate reader was purchased from Tecan Company in Switzerland. Compound 6 was provided by Suzhou Manyuan Biotechnology Co., Ltd., and sodium pentobarbital and Blebbistatin (Ble) were purchased from Sigma Company in the United States.

[0027] 2. Experimental Animals

[0028] Male C57BL / 6J mice, 6 - 8 weeks old, weighing 20 - 24 g, SPF grade, were purchased from the Comparative Medicine Center of Yangzhou University and met the quality standards of ordinary laboratory animals. License number: SCXK (Jiangsu) 2020 - 0019. The mice were housed separately in SPF - level individually ventilated cages (IVC). The temperature of the breeding room was 23 - 25 °C, and the relative humidity was 40% - 80%. After 3 - 5 days of adaptive breeding, animal experiments were started.

[0029] II. Experimental methods

[0030] 1. Grouping of experimental animals and administration

[0031] Thirty male C57BL / 6J mice were randomly divided into 6 groups (6 mice / group):

[0032] Sham - operation group: Intraperitoneally injected with an equal volume of normal saline 0.5 h before surgery, once.

[0033] Model group: Intraperitoneally injected with an equal volume of normal saline 0.5 h before surgery, once.

[0034] Low - dose group of Compound 6: Intraperitoneally injected with Compound 6 at 0.3 mg / kg 0.5 h before surgery, once.

[0035] Medium - dose group of Compound 6: Intraperitoneally injected with Compound 6 at 1 mg / kg 0.5 h before surgery, once.

[0036] High - dose group of Compound 6: Intraperitoneally injected with Compound 6 at 3 mg / kg 0.5 h before surgery, once.

[0037] Ble group: Intraperitoneally injected with Ble at 3 mg / kg 0.5 h before surgery, once.

[0038] 2. Preparation of carotid artery thrombosis model in mice by ferric chloride method

[0039] C57BL / 6J mice were anesthetized intraperitoneally with pentobarbital according to body weight. After verifying the absence of the righting reflex, the animals were placed in the supine position, and their four limbs were fixed on the operating board. After shaving the hair on the neck, a 1 - cm midline incision was made, and the right tympanic gland of the mouse was separated and turned outward. The pulsation of the common carotid artery could be seen on the medial side of the triangle formed by the left omohyoid muscle, sternohyoid muscle, and digastric muscle of the mouse. The right common carotid artery was separated and exposed. A filter paper of about 1 cm × 0.5 cm was placed under it, and a filter paper of about 0.5 cm × 0.2 cm dipped in 5% ferric chloride was attached above it for 3 min. Then the two filter papers were discarded, and the carotid artery was carefully rinsed with PBS. The laser speckle Doppler flow imaging system was used to detect the carotid blood flow to determine whether the model was successfully established.

[0040] 3. Collection, embedding, and sectioning of carotid arteries

[0041] The blood flow value was measured on a blood flow meter to be less than 200 PU and maintained for ≥10 min, or after 0.5 h of measurement, the mice were sacrificed, fixed on a flat plate, and the common carotid artery was carefully isolated. During the isolation process, try to avoid stretching the blood vessel. After rinsing with PBS, the water was blotted dry with filter paper and then placed on a balance to weigh the wet weight of the thrombus. The length of the thrombus was measured with a vernier caliper and placed in a centrifuge tube containing 4% paraformaldehyde and fixed at 4°C for 24 h. After fixation, the tissue was transferred to a 40% sucrose solution and dehydrated at 4°C until the tissue sank to the bottom. After dehydration, it was embedded in OCT, and frozen sections of 10 μm / slice were cut using a cryostat. The sections were attached to adhesive slides with a surface treated with polylysine using the direct attachment method, stored at -70°C, and prepared for staining.

[0042] 4. Statistical analysis

[0043] All data were statistically analyzed using Graphpad Prism 8 software, expressed as mean±SEM. For comparisons between two groups, Students’t test was used. When comparing three groups or more, one-way ANOVA was used, and Dunnett’s test was used for the test. P<0.05 was considered statistically significant.

[0044] III. Experimental results

[0045] As Figure 1 shown, compared with the sham operation group, thrombosis in the model group was significantly increased ( ### P<0.001); compared with the model group, compound No. 6 at 3 mg / kg showed significant differences ( *** P<0.001), which could significantly inhibit the formation of arterial thrombus. At 0.3 mg / kg and 1 mg / kg, there were no significant differences compared with the model group. In summary, the treatment of compound No. 6 for arterial thrombus was dose-dependent, and the treatment effect was optimal at 3 mg / kg. As Figure 1 shown in C below, the H&E results also showed that compound No. 6 (3 mg / kg) could inhibit the formation of arterial thrombus and reduce the generation of blood clots in blood vessels.

[0046] Example 2

[0047] Effect of different doses of compound No. 6 on the expression of tissue factor in the carotid artery thrombus tissue of mice induced by ferric chloride

[0048] I. Experimental materials

[0049] The instruments, reagents, and experimental animals were the same as those in Example 1.

[0050] II. Experimental methods

[0051] 1. Animal grouping, model establishment, and drug administration were the same as those in Example 1

[0052] 2. Tissue Immunofluorescence Staining

[0053] Take out the glass slides stored at -20°C and equilibrate them at room temperature for 30 min. Immerse them in PBS for 5 min to remove OCT. Blot dry the liquid droplets on the glass slides, use a brush to circle the sample, add the blocking solution dropwise inside the circle, and block at room temperature for 1 h. After blotting dry the blocking solution, rinse with PBS 3 times, 5 min each time. Add the diluted primary antibody and incubate overnight at 4°C. Blot dry the primary antibody, rinse with PBS 3 times, 5 min each time, add the diluted fluorescently labeled secondary antibody, and incubate at room temperature for 2 h. Rinse with PBS 3 times, 5 min each time, blot dry the liquid, add DAPI, and let it stand at room temperature for 5 min. Rinse with PBS 1 time, 5 min each time, blot dry, add the anti-fluorescence quencher to mount the slides, and take pictures under a laser confocal microscope.

[0054] 3. Data processing is the same as in Example 1.

[0055] III. Experimental Results

[0056] As Figure 2 shown, immunohistochemistry and immunofluorescence results showed that compared with the sham operation group, the expression of tissue factor (TF) in the carotid artery thrombus tissue of the model group mice was significantly increased, and compound No. 6 (3 mg / kg) could significantly reduce the expression of TF in the carotid artery thrombus tissue of mice induced by ferric chloride ( * P<0.05), inhibiting thrombus formation.

[0057] Example 3

[0058] Effects of Different Doses of Compound No. 6 on the Contents of Coagulation Factors and Inflammatory Factors in Plasma of Mice with Carotid Artery Thrombosis Induced by Ferric Chloride

[0059] I. Experimental Materials

[0060] The instruments and experimental animals are the same as in Example 1. Kits for 6-keto-prostaglandin F1α (6-keto-PGF1α), thromboxane B2 (TXB 2 ) and tissue factor (TF) were purchased from Nanjing Jinyibai Biotechnology Co., Ltd.

[0061] II. Experimental Methods

[0062] 1. Animal grouping, modeling, and drug administration are the same as in Example 1.

[0063] 2. Determination of the Contents of Coagulation Factors and Inflammatory Factors in Mouse Plasma

[0064] The determination of the contents of 6-keto-prostaglandin F1α (6-keto-PGF1α), thromboxane B2 (TXB2) and tissue factor (TF) in mouse plasma was carried out according to the kit instructions.

[0065] 3. The data processing is the same as that in Example 1.

[0066] III. Experimental Results

[0067] The results are as Figure 3 shown. Compared with the sham operation group, the levels of TXB 2 and TF in the plasma of mice in the model group were significantly increased, while the level of 6-keto-PGF1α was decreased. After administration of Compound No. 6, it was observed that the levels of TF and TXB 2 in the plasma of mice were decreased, while the level of 6-keto-PGF1α was increased. The above results indicate that Compound No. 6 can inhibit platelet activation and inflammatory response in carotid artery thrombosis induced by FeCl 3 .

Claims

1. Use of the compound shown in the following formula in the preparation of antithrombotic drugs: 。 2. The use according to claim 1, characterized in that: The drug reduces tissue factor expression.

3. The use according to claim 1, characterized in that: The drug reduces blood clot formation.

4. The use according to claim 1, characterized in that: The drug regulates the expression of inflammatory factors.

5. Use of a pharmaceutical composition in the preparation of an antithrombotic drug, characterized in that: The pharmaceutical composition comprises an active ingredient, and the active ingredient is a compound shown in the following formula: 。 6. The use according to claim 5, characterized in that: The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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