Anticoagulant composition as well as preparation method, related product and application thereof

Abstract

The invention provides an anticoagulant composition as well as a preparation method, a related product and application thereof. Belongs to the technical field of medicine. The anticoagulant composition comprises hydroxysafflor yellow A and dehydrated safflor yellow B in a mass ratio of (0.5-0.75): 1. The anticoagulant composition provided by the invention can synergistically prolong APTT, inhibit FXa activity and inhibit zebra fish thrombosis, and is expected to develop drugs with better curative effects for treating or preventing cardiovascular diseases, cerebrovascular diseases, cerebral apoplexy, venous thromboembolic diseases and blood system diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an anticoagulant composition, a preparation method thereof, related products and uses. BACKGROUND

[0002] Anticoagulant therapy is a core strategy for preventing or treating thrombotic diseases, and its indications widely cover cardiovascular diseases and venous thromboembolic diseases. First, in the field of cardiovascular diseases, atrial fibrillation (AF) is one of the most common indications, because blood stasis in the left atrium of patients with atrial fibrillation is prone to thrombosis, which significantly increases the risk of stroke, and long-term anticoagulant therapy such as rivaroxaban or warfarin is needed. In addition, patients receiving mechanical heart valve replacement usually need to use warfarin for anticoagulation for life to prevent artificial valve-related thrombosis. Second, venous thromboembolic diseases are another focus of anticoagulant therapy, including deep vein thrombosis (DVT) and pulmonary embolism (PE). Patients with DVT can prevent thrombus extension and shedding leading to fatal PE through anticoagulant therapy, and patients with PE need long-term anticoagulation with drugs such as apixaban or enoxaparin to prevent recurrence. Finally, some blood system diseases also require individualized anticoagulant intervention, such as patients with genetic thrombophilia (such as coagulation factor V Leiden mutation) who need lifelong prophylactic anticoagulation due to congenital hypercoagulable state.

[0003] The currently used anticoagulants are mainly divided into the following four categories: vitamin K antagonists, heparin drugs, direct thrombin inhibitors and Xa factor inhibitors. Vitamin K antagonists such as warfarin exert an anticoagulant effect by inhibiting the synthesis of vitamin K-dependent coagulation factors (II, VII, IX, X), and are suitable for long-term anticoagulation needs such as atrial fibrillation and heart valve disease. However, due to its strong anticoagulant effect, it also has a high incidence of bleeding. Heparin drugs such as ordinary heparin and low molecular weight heparin act by activating antithrombin III (ATIII) to inhibit thrombin (IIa) and Xa factor. The bleeding rate of ordinary heparin is also high, although the bleeding risk of low molecular weight heparin is lower, but gastrointestinal bleeding or postoperative bleeding may still occur. Direct thrombin inhibitors such as dabigatran and argatroban and Xa factor inhibitors such as rivaroxaban also have adverse reactions such as gastrointestinal symptoms, liver and kidney function damage, etc.

[0004] Safflower is a plant of the family Asteraceae Carthamus tinctoriusThe dried flowers of L. are commonly used blood-activating and stasis-removing drugs, and are often used to treat cardiovascular and cerebrovascular diseases such as coronary heart disease and cerebral thrombosis. Hydroxysafflor Yellow A (HSYA) is one of the main active ingredients of safflower, and its structural formula is as follows. Xia Yuye et al. disclosed in the article "Effects of Hydroxysafflor Yellow A on Thrombosis and Platelet Aggregation in Rats" (Chinese Pharmacological Bulletin, 2005, 21(11): 1400~1401) that intravenous injection of 1~4 mg / kg of hydroxysafflor yellow A (HSYA) has a significant inhibitory effect on thrombosis and arachidonic acid (AA)-induced platelet aggregation, alleviating post-ischemic hypoperfusion, platelet microthrombosis, and cerebral circulation disorders that may be caused by the release of AA.

[0005] Anhydrosafflor yellow B (AHSYB) is also an important chalcone active ingredient in safflower. Its structural formula is shown below. It and hydroxysafflor yellow A (HSYA) are both major active components of safflower.

[0006] The chemical structure of hydroxysafflor yellow A is as follows: .

[0007] The chemical structure of dehydrated safflower yellow B is as follows: .

[0008] Chinese invention patent application CN105687282A discloses a safflower anti-Parkinson's disease effective fraction drop pill. The pill is made from an extract of the safflower anti-Parkinson's disease effective fraction and a matrix. The extract contains hydroxysafflower yellow A and dehydrated safflower yellow B. However, the pill's effect is primarily anti-Parkinson's disease and has no anticoagulant effect.

[0009] Given the numerous side effects of the aforementioned anticoagulant drugs, developing anticoagulant drugs with significant efficacy and high safety is of great clinical significance. Therefore, there is an urgent need to provide an anticoagulant composition with significant efficacy. Summary of the Invention

[0010] In view of this, the object of the present invention is to provide an anticoagulant composition with significant therapeutic effect. Compared with the prior art, the composition of the present invention has significant therapeutic effect.

[0011] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides an anticoagulant composition comprising hydroxysafflor yellow A and dehydrated safflor yellow B in a mass ratio of 0.5-0.75:1.

[0012] As a specific embodiment of the present invention, the anticoagulant composition comprises hydroxysafflor yellow A and dehydrated safflor yellow B in a mass ratio of 0.586:1.

[0013] In another aspect, the present invention provides an anticoagulant drug, the active ingredient of which includes the anticoagulant composition.

[0014] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0015] The pharmaceutical carrier used can be solid, liquid or gas. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil and water.

[0016] When preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, ethanol, oils, alcohols, flavoring agents, preservatives, colorants, and the like can be used to form oral liquid formulations such as suspensions, elixirs, and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, and disintegrants can be used to form oral solid formulations such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or nonaqueous techniques.

[0017] Tablets containing the anticoagulant composition of the present invention can be prepared by tableting or molding, optionally with the use of one or more auxiliary ingredients or adjuvants. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as a powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersant. Molded tablets can be molded in a suitable machine, i.e., a powdered compound mixture moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to about 5 g of active drug, mixed with an appropriate and convenient carrier material, which may constitute about 5% to 95% of the total composition. Unit dosage forms typically contain about 1 mg to about 2 g of active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0018] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, and preservatives (including antioxidants). In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, colorants, and flavoring agents, may be added. The preparation is made isotonic with the blood of the intended recipient. Components containing the anticoagulant composition of the present invention can also be prepared in the form of a powder or concentrate.

[0019] Preferably, the dosage form of the drug is selected from powder, pill, capsule, granule, tablet or oral solution.

[0020] Furthermore, the pharmaceutically acceptable excipients include one or more of solvents, buffers, fillers, binders, wetting agents, absorption enhancers and surfactants.

[0021] A third aspect of the present invention provides an anticoagulant solution, comprising the anticoagulant composition and a pharmaceutically acceptable solvent.

[0022] Furthermore, the total concentration of hydroxysafflor yellow A and dehydrated safflor yellow B in the anticoagulant solution is 9-12 g / L. Furthermore, in the anticoagulant solution, the concentration of hydroxysafflor yellow A is 3.136-4.70 g / L, and the concentration of dehydrated safflor yellow B is 6.27 g / L.

[0023] The fourth aspect of the present invention provides a method for preparing the anticoagulant solution, which comprises the following steps: taking a formula amount of hydroxysafflor yellow A and dehydrated safflor yellow B, and mixing them with a solvent.

[0024] The fifth aspect of the present invention provides an application of the anticoagulant composition, the anticoagulant drug or the anticoagulant solution in the preparation of drugs for treating or preventing cardiovascular diseases, cerebrovascular diseases, stroke, venous thromboembolic diseases and blood system diseases.

[0025] Furthermore, the cardiovascular disease is selected from one or more of atrial fibrillation, coronary heart disease, myocardial infarction, valvular heart disease, left ventricular thrombosis or chronic heart failure.

[0026] Furthermore, the cerebrovascular disease is selected from one or more of cerebral atherosclerosis, cerebral thrombosis or ischemic cerebral infarction.

[0027] Furthermore, the venous thromboembolic disease is deep vein thrombosis and / or pulmonary embolism.

[0028] Furthermore, the blood system disease is thrombophilia and / or disseminated intravascular coagulation.

[0029] Compared with the prior art, the present invention has the following beneficial effects: In the APTT assay, a composition of hydroxysafflor yellow A and dehydrosafflor yellow B with a mass ratio of 0.5-0.75:1 showed a synergistic effect in prolonging the APTT, with a mass ratio of 0.586:1 being optimal. In the FXa inhibitory activity assay, a composition of hydroxysafflor yellow A and dehydrosafflor yellow B with a mass ratio of 0.586:1 exhibited a synergistic inhibitory effect on FXa activity compared to both hydroxysafflor yellow A and dehydrosafflor yellow B alone. Furthermore, a composition of hydroxysafflor yellow A and dehydrosafflor yellow B with a mass ratio of 0.586:1 also demonstrated a synergistic effect in inhibiting zebrafish thrombosis.

[0030] In summary, the anticoagulant composition comprising hydroxysafflor yellow A and dehydrated safflor yellow B provided by the present invention can synergistically prolong APTT, inhibit FXa activity, and inhibit thrombosis in zebrafish, and is expected to develop more effective drugs for the treatment or prevention of cardiovascular diseases, venous thromboembolic diseases, and blood system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the results of the determination of the FXa activity inhibition by the anticoagulant composition provided in Example 1; Figure 2 Graphs showing the experimental results of the anticoagulant composition provided in Example 1 for inhibiting thrombosis in zebrafish; (a) is a fluorescence micrograph of zebrafish, and (b) is a bar graph showing the percentage of thrombosis in zebrafish embryos to that in all zebrafish embryos. DETAILED DESCRIPTION

[0032] Terms and Claims of the Present Invention: 1. The articles "a", "an" and "the" include plural referents unless expressly limited to one or more referents otherwise.

[0033] 2. Numerical ranges: Unless expressly stated otherwise, all ranges or ratios disclosed herein are to be understood to include any and all subranges or subratios contained therein. For example, a range or ratio stated as 1 to 30 is to be considered inclusive of any and all subranges or subratios, integers, decimals, or subranges or subratios comprised therein, between a minimum of 1 and a maximum of 30, including any subranges or subratios, integers, decimals, or subranges or subratios comprised therein.

[0034] 3. As used herein, the terms "comprises", "includes", "has", "may", "contain" and their variations are open-ended conjunctions or terms that do not exclude the possibility of additional compositions or structures.

[0035] The following non-limiting examples can make the ordinary skilled in the art more comprehensive understanding of the present application, but not in any way limit the present application. The following content is only an exemplary description of the scope of the present application, those skilled in the art can make various changes and modifications to the present application according to the disclosed content, and it should also belong to the scope of the present application claimed.

[0036] The present application is further described below in the manner of specific examples. The various chemical reagents used in the embodiments of the present application are obtained by conventional commercial routes unless otherwise specified. If not otherwise specified, the content described below is the mass content. If not otherwise specified, it is understood to be carried out at room temperature.

[0037] Example 1 An anticoagulant composition consisting of hydroxysafflor yellow A and anhydrosafflor yellow B in a mass ratio of 0.586:1.

[0038] The anticoagulant composition can be made into an anticoagulant solution (mixed aqueous solution of hydroxysafflor yellow A and anhydrosafflor yellow B). The concentration of hydroxysafflor yellow A is 3.675 g / L, and the concentration of anhydrosafflor yellow B is 6.27 g / L.

[0039] Example 2 An anticoagulant composition consisting of hydroxysafflor yellow A and anhydrosafflor yellow B in a mass ratio of 0.5:1.

[0040] The anticoagulant composition can be made into an anticoagulant solution (mixed aqueous solution of hydroxysafflor yellow A and anhydrosafflor yellow B). The concentration of hydroxysafflor yellow A is 3.1362 g / L, and the concentration of anhydrosafflor yellow B is 6.27 g / L.

[0041] Example 3 An anticoagulant composition consisting of hydroxysafflor yellow A and anhydrosafflor yellow B in a mass ratio of 0.75:1.

[0042] The anticoagulant composition can be made into an anticoagulant solution (mixed aqueous solution of hydroxysafflor yellow A and anhydrosafflor yellow B). The concentration of hydroxysafflor yellow A solution is 4.704 g / L and the concentration of anhydrosafflor yellow B solution after dilution is 6.27 g / L.

[0043] Comparative Example 1 An anticoagulant composition consisting of hydroxysafflor yellow A and anhydrosafflor yellow B in a mass ratio of 0.8:1.

[0044] The anticoagulant composition can be prepared into an anticoagulant solution (a mixed aqueous solution of hydroxysafflor yellow A and dehydrated safflor yellow B), wherein the concentration of the hydroxysafflor yellow A solution is 5.016 g / L and the concentration of the diluted dehydrated safflor yellow B solution is 6.27 g / L.

[0045] Comparative Example 2 An anticoagulant composition consists of hydroxysafflower yellow A and dehydrated safflower yellow B in a mass ratio of 0.45:1.

[0046] The anticoagulant composition can be prepared into an anticoagulant solution (a mixed aqueous solution of hydroxysafflor yellow A and dehydrated safflor yellow B), wherein the concentration of the hydroxysafflor yellow A solution is 3.087 g / L and the concentration of the diluted dehydrated safflor yellow B solution is 6.858 g / L.

[0047] The anticoagulant solutions in Examples 1 to 3 and Comparative Examples 1 and 2 were prepared by the following preparation method: A certain amount of hydroxysafflor yellow A and anhydrosafflor yellow B are respectively dissolved in a small amount of DMSO to obtain a mother solution of hydroxysafflor yellow A and a mother solution of anhydrosafflor yellow B. The two mother solutions are mixed with water in a certain proportion to obtain an anticoagulant solution.

[0048] Test example: Verification of synergistic effect in the following experiments: Calculate the expected additive effect and CI (drug synergy coefficient) by calculating formulas (1) and (2).

[0049] Expected Additive Effect = E A +E B -E A ×E B (1) CI=(E A +E B -E A ×E B ) / E AB (2) Among them, E A represents the effect of hydroxysafflor yellow A, E B represents the effect of dehydrated safflower yellow B, E AB Represents the effect of the anticoagulant composition; if CI is less than 1, that is, E AB The result is greater than the expected additive effect, which proves that hydroxysafflower yellow A and dehydrated safflower yellow B have synergistic effects. A , E B and E AB The blank control group is deducted as 1 for calculation. When calculating the expected additive effect, it is necessary toA , E B and E AB Subtract the value of the blank control group from 1 and add 1 to the result.

[0050] 1. APTT (activated partial thromboplastin time) test Mix 10 μL of the test solution with 40 μL of sheep plasma, then add 50 μL of preheated APTT reagent. After incubation at 37°C for 3 minutes, add 50 μL of preheated CaCl₂ solution (0.025 M), and record the clotting time. When the test solution is water, the measured time is considered the blank control. All other APTT values ​​are expressed relative to the blank control as 1.

[0051] Table 1

[0052] The test results are shown in Table 2 below (n=3, mean ± standard deviation, relative value with the blank control group as 1).

[0053] Table 2

[0054] The results showed that hydroxysafflor yellow A and dehydrated safflor yellow B had a synergistic effect in prolonging APTT. In addition, the CI values ​​of Examples 1 to 3 were greater than those of Comparative Examples 1 and 2.

[0055] 2. Determination of FXa inhibitory activity The experiment was carried out in four groups.

[0056] Panel 1: Add 50 μL of the test solution to each well of a 96-well plate. Then, add 50 μL of FXa (25 nM) solution to each well and mix thoroughly. Incubate the plate in a thermomixer at 37°C, 300 rpm for 30 minutes. Then, add 50 μL of the substrate S-2765 (0.5 mg / mL) to each well. Incubate at 37°C, 300 rpm for 10 minutes. Analyze the test solution at 405 nm using a microplate reader and record the absorbance of each well.

[0057] Group 2: Add 50 μL of the test solution to each well of a 96-well plate. Then, add 50 μL of PBS buffer to each well and mix thoroughly. Incubate the plate in a thermomixer at 37°C, 300 rpm for 30 minutes. Then, add 50 μL of the substrate S-2765 (0.5 mg / mL) to each well. Incubate at 37°C, 300 rpm for 10 minutes. Analyze the test solution at 405 nm using a microplate reader and record the absorbance of each well.

[0058] The third group: 50 μL PBS buffer was added to each well of the 96-well plate, and then 50 μL of FXa (25 nM) solution was added to each well, and mixed. After incubation at 37 °C, 300 r / min for 30 min in a constant temperature mixer, 50 μL of substrate S-2765 (0.5 mg / mL) was added to each well. After incubation at 37 °C, 300 r / min for 10 min, the absorbance value of each well was detected and recorded at 405 nm using a microplate reader.

[0059] The fourth group: 100 μL PBS buffer was added to each well of the 96-well plate, and then incubated at 37 °C, 300 r / min for 30 min in a constant temperature mixer. Then 50 μL of substrate S-2765 (0.5 mg / mL) was added to each well. After incubation at 37 °C, 300 r / min for 10 min, the absorbance value of each well was detected and recorded at 405 nm using a microplate reader.

[0060] The inhibition rate of the test solution was calculated according to the inhibition rate calculation formula (3).

[0061] Inhibition rate E (%) = [1-(A1-A2) / (A3-A4)] x 100% (3) Wherein, A 1- A4 is the average absorbance value measured by the first group to the fourth group, respectively.

[0062] The compositions of the test solutions of Example 1-Example 3 and Comparative Example 1-Comparative Example 2 were 20 times diluted solutions shown in Table 1.

[0063] The inhibition rate detection results are shown in Table 3 and Figure 1 E A , E B and E AB are expressed as mean ± standard deviation. The results show that the anti-coagulation mixture containing hydroxysafflor yellow A and anhydrosafflor yellow B has much higher inhibition activity on FXa than the single component, and hydroxysafflor yellow A and anhydrosafflor yellow B can synergistically inhibit the activity of FXa. Moreover, the CI values of Example 1-Example 3 are greater than those of Comparative Example 1 and Comparative Example 2.

[0064] Table 3

[0065] 3. Inhibition of thrombosis in zebrafish Transgenic zebrafish Tg(LCR:eGFP)Embryos were collected and placed in culture dishes. E3 medium containing methylene blue (60× stock solution: NaCl 17.4 g / L, KCl 0.8 g / L, CaCl2·2H2O 2.9 g / L, MgCl2·6H2O 4.89 g / L) was added. Twelve hours later, E3 medium containing 200 μM 1-phenyl-2-thiourea (PTU) was added to inhibit melanin production and facilitate subsequent observations. At 56 hpf, embryos were transferred to 24-well plates, with eight embryos per well. After aspirating the liquid from the wells, 1 mL of E3 medium containing PTU (control group and modeling group), aspirin solution with a final concentration of 10 μg / mL (positive drug group), hydroxysafflor yellow A solution with a final concentration of 61.25 mg / L, dehydrated safflor yellow B solution with a final concentration of 104.49 mg / L, and an anticoagulant solution diluted 60-fold to the final concentration of Example 1 were added. If embryo membranes were not ruptured, proteinase K was used to remove the membranes. After 24 hours of pre-protection with drug administration, the embryos were rinsed three times with E3 medium containing PTU. Then, E3 medium containing PTU was added to the control group, while 2 μM phenylhydrazine (PHZ) was added to the modeling group, positive drug group, and drug administration group to establish the model. After 14 hours of modeling stimulation, the embryos were anesthetized with anesthetic and transferred, one per well, to the lid of a 24-well plate. Images were captured automatically with a Leica camera at 100 frames / second. Following imaging, the same part of each zebrafish embryo was selected for observation. According to the severity of zebrafish thrombosis, they were divided into normal, mild, and severe. Figure 2 . Figure 2 In the above, A represents hydroxysafflower yellow A, B represents dehydrated safflower yellow B, and C represents the anticoagulant solution provided in Example 1.

[0066] Depend on Figure 2 It can be seen that the proportion of normal zebrafish after administration of hydroxysafflor yellow A and dehydrosafflor yellow B alone is less than the proportion of normal zebrafish after administration of the anticoagulant solution of Example 1. This indicates that the anticoagulant solution containing hydroxysafflor yellow A and dehydrosafflor yellow B has a synergistic effect in inhibiting thrombosis in zebrafish.

[0067] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An anticoagulant composition, characterized in that The anticoagulant composition comprises hydroxysafflower yellow A and dehydrated safflower yellow B in a mass ratio of 0.5-0.75:

1.

2. The anticoagulant composition according to claim 1, characterized in that The anticoagulant composition comprises hydroxysafflower yellow A and dehydrated safflower yellow B in a mass ratio of 0.586:

1.

3. An anticoagulant drug, characterized in that: The active ingredient comprises the anticoagulant composition according to claim 1 or 2.

4. An anticoagulant solution, characterized in that The invention comprises the anticoagulant composition according to claim 1 or claim 2 and a pharmaceutically acceptable solvent.

5. The anticoagulant solution according to claim 4, characterized in that The total concentration of hydroxysafflor yellow A and anhydrosafflor yellow B in the anticoagulant solution is 9-12 g / L.

6. The method for preparing the anticoagulant solution according to any one of claims 4 to 5, characterized in that: The method comprises the following steps: taking a formula amount of hydroxy safflower yellow pigment A and dehydrated safflower yellow pigment B, and mixing them with a solvent to obtain the pigment.

7. Use of the anticoagulant composition according to claim 1 or 2, the anticoagulant drug according to claim 3, or the anticoagulant solution according to any one of claims 4 to 5 in the preparation of drugs for treating or preventing cardiovascular diseases, cerebrovascular diseases, stroke, venous thromboembolic diseases and blood system diseases.

8. The use according to claim 7, characterized in that The cardiovascular disease is selected from one or more of atrial fibrillation, coronary heart disease, myocardial infarction, valvular heart disease, left ventricular thrombosis or chronic heart failure.

9. The use according to claim 7, characterized in that The venous thromboembolic disease is deep vein thrombosis and / or pulmonary embolism.

10. The use according to claim 7, characterized in that The blood system disease is thrombophilia and / or disseminated intravascular coagulation.

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