Application of cytoskeleton regulator in preparation of coagulant drug

Abstract

The invention relates to application of a cytoskeleton regulator in preparation of a blood coagulation promoting drug, and belongs to the technical field of biological medicines, and the blood coagulation promoting drug is a drug for treating or preventing hemorrhagic diseases related to low blood coagulation function. The hemorrhagic diseases comprise traumatic hemorrhage, surgical operation related hemorrhage, hepatopathy related hemorrhage, postpartum hemorrhage, hemophilia, hemorrhage caused by excessive anticoagulant drugs, thrombocytopenia or dysfunction related hemorrhage; the cytoskeleton regulator is one or more of a compound for inhibiting polymerization of actin microfilaments, a compound for inhibiting shrinkage of myosin and a ROCK kinase inhibitor; the cytoskeleton regulator can be used for preparing a procoagulant treatment drug, the effect is played by inducing endogenous cells of a body to release procoagulant particles, a brand new strategy is provided for a clinical hemostatic drug, and the cytoskeleton regulator can remarkably shorten the coagulation time of whole blood; the bleeding time can be remarkably shortened, the total blood loss amount is reduced, and the powerful in-vivo bleeding stopping effect is achieved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of cytoskeleton regulators in the preparation of procoagulant drugs. Background Technology

[0002] Blood clotting is a crucial physiological process for preventing blood loss after vascular injury. Rapid and effective hemostasis is essential in clinical situations such as severe trauma, major surgery, disseminated intravascular coagulation (DIC), anticoagulant overdose, and congenital coagulation disorders such as hemophilia.

[0003] Currently, drugs used clinically to promote coagulation mainly include: 1) supplementary coagulation factors, such as recombinant activated human coagulation factor VII (rFVIIa), fibrinogen concentrate, and prothrombin complex concentrate. Although these drugs are directly effective, they generally suffer from high cost, short half-life, potential immunogenicity, and the risk of inducing thrombosis; 2) antifibrinolytic drugs, such as tranexamic acid and aminocaproic acid. They stabilize existing thrombi by inhibiting plasmin activity, but their effectiveness is limited for patients with slow coagulation initiation or insufficient thrombus formation capacity. Therefore, developing a drug with a novel mechanism of action that can efficiently utilize and enhance the body's own coagulation potential is a pressing technical problem to be solved in this field.

[0004] Microparticles (MPs), particularly those derived from platelets and megakaryocytes, have become a research hotspot in recent years. These are vesicle-like structures shed from the cell membrane during cell activation or apoptosis. Studies have shown that the outer membrane of these microparticles exposes negatively charged phosphatidylserine residues, providing a crucial catalytic surface for coagulation complexes (such as prothrombinase complex and endogenous factor ten enzyme complex), thereby significantly accelerating thrombin formation—a process known as "providing additional coagulation surface." Furthermore, these microparticles can carry and release various procoagulant substances, such as platelet factor IV, von Willebrand factor, and tissue factor, further enhancing the coagulation response. Research has found that circulating microparticles mainly originate from platelet microparticles, leukocytes, and endothelial cells. Platelet-derived MPs are the most abundant, accounting for approximately 70% to 90% of total circulating MPs, while leukocyte-derived MPs account for less than 10%, and endothelial-derived MPs account for a smaller proportion, approximately 5% to 15%. These microparticles (MPs) are lipid bilayer vesicles carrying cytoplasmic material and surface proteins from their parent cells, playing roles in intercellular communication, inflammation, and thrombosis. New research has found that platelet MPs in peripheral circulation are actually primarily derived from megakaryocyte microparticles from their parent cells; therefore, regulating the release of megakaryocyte and platelet microparticles could potentially significantly influence the total amount of cellular microparticles in the bloodstream.

[0005] The cytoskeleton, mainly composed of microtubules, microfilaments (actin filaments), and intermediate filaments, is a key structure for maintaining cell morphology and participating in substance transport and signal transduction. Dynamic changes in the cell membrane, including budding, vesicle formation, and microparticle release, are precisely and dynamically regulated by the cytoskeleton. For example, the depolymerization and remodeling of the actin network is considered one of the direct driving forces behind cell membrane budding and microparticle release.

[0006] Cytoskeleton modulators are a class of drugs widely used in other therapeutic areas. For example, microtubule modulators such as paclitaxel and vincristine, and microfilament modulators such as cytochalasin, are primarily used as anti-tumor drugs due to their cell division-inhibiting properties. In recent years, some small molecule compounds involved in cytoskeleton signaling pathways, such as the Rho / ROCK pathway, have also been used to regulate the microfilament cytoskeleton. However, existing technologies have never disclosed or revealed the use of these cytoskeleton modulators for procoagulant therapy, or for achieving rapid hemostasis by actively inducing cells (especially megakaryocytes or platelets) to release functional procoagulant microparticles. These applications are innovative both conceptually and technologically. Summary of the Invention

[0007] Technical problem solved: This invention provides the application of cytoskeleton regulators in the preparation of procoagulant drugs, which solves the technical defects of existing procoagulant drugs, such as single mechanism of action, high price, short half-life, possible immunogenicity, and risk of inducing thrombosis.

[0008] Technical solution: Application of cytoskeleton regulators in the preparation of procoagulant drugs.

[0009] Furthermore, the procoagulant is a drug used to treat or prevent bleeding disorders associated with impaired coagulation function.

[0010] Furthermore, the bleeding disorders include traumatic bleeding, surgical bleeding, liver disease-related bleeding, postpartum hemorrhage, hemophilia, bleeding caused by anticoagulant overdose, and bleeding related to thrombocytopenia or functional disorders.

[0011] Furthermore, the cytoskeleton regulator is a small molecule compound that targets and regulates cytoskeleton signaling pathways.

[0012] Furthermore, the cytoskeleton regulator is one or more of the following: compounds that inhibit actin microfilament polymerization, compounds that inhibit myosin contraction, and ROCK kinase inhibitors.

[0013] Furthermore, the compound that inhibits actin microfilament polymerization is cytochalasin D and / or the LIMK1 / 2 inhibitor BMS-5.

[0014] Furthermore, the compound that inhibits myosin contraction is the ATPase inhibitor Blebbistatin and / or the myosin light chain kinase inhibitor ML-7.

[0015] Furthermore, the ROCK kinase inhibitor is Y-39983 and / or Y-27632.

[0016] Furthermore, the procoagulant drug includes cytoskeleton regulators and pharmaceutically acceptable excipients.

[0017] Furthermore, the excipients are one or more of the following: fillers, stabilizers, diluents, adjuvants, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.

[0018] Beneficial effects: 1. This application uses known cytoskeleton regulators as active ingredients to prepare drugs that promote blood coagulation and to prepare pharmaceutical compositions for treating hemorrhagic diseases; 2. Novel Mechanism of Action: This invention reveals for the first time that various cytoskeleton regulators can be used to prepare procoagulant therapeutic drugs, which exert their effects by inducing endogenous cells (such as megakaryocytes / platelets) to release procoagulant microparticles, providing a novel strategy for clinical hemostatic drugs; 3. Clear hemostatic effect: In vitro experimental data confirm that the various cytoskeleton regulators used in this invention can significantly shorten the clotting time of whole blood; in vivo animal models (such as the mouse tail tip hemorrhage model) also prove that the application of the drug of this invention can significantly shorten the bleeding time and reduce the total blood loss, and has a strong in vivo hemostatic effect. 4. Potential broad-spectrum applicability: Since this invention utilizes a universal mechanism to enhance the body's own coagulation surface, it may have therapeutic potential for bleeding caused by a variety of reasons (such as lack of specific coagulation factors or platelet dysfunction). 5. Wide range of drug sources: The cytoskeleton regulators involved in this invention are mostly mature small molecule drugs that are already on the market. Their chemical properties are stable and their pharmacological effects have been extensively studied, which can potentially shorten the new drug development cycle and reduce costs. Attached Figure Description

[0019] Figure 1 This is a diagram showing the cytoskeleton regulators that promote the release of megakaryocyte microparticles in Example 1 of this invention, *compared to the control group (t-test). P <0.05, & compared with similar drugs, P <0.05; Figure 2 This is a graph showing the effect of different concentrations of cytoskeleton regulators on megakaryocyte microparticle release in Example 1 of the present invention, with intergroup comparisons (ONE-ANOVA). * P<0.05, ** P <0.01, *** P <0.001; Figure 3 A diagram illustrating the possible mechanism by which the cytoskeleton regulator promotes the release of megakaryocyte microparticles in Example 1 of this invention; Figure 4 This is a diagram illustrating the rapid in vivo promotion of cellular microparticle release by the cytoskeleton regulator in Example 3 of the present invention. Figure 5 The graphs show the acceleration of hemostasis by the cytoskeleton regulator in Example 3 of the present invention, where Figure A represents the total bleeding time of the mice and Figure B represents the total blood loss of the mice. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be noted that the embodiments described herein are for illustrative purposes only and do not constitute any limitation on the scope of the invention.

[0021] Example 1, such as Figure 1 As shown, the application of cytoskeleton regulators in the preparation of procoagulant drugs and the verification of the effect of cytoskeleton regulators in inducing the release of procoagulant microparticles from megakaryocytes are demonstrated. To verify the mechanism of action of the drug described in this invention, this embodiment tested the effect of the drug on the release of procoagulant microparticles from human megakaryocyte cell line Meg-01.

[0022] Experimental methods: Step 1: Screening for cytoskeleton regulators that promote the release of procoagulant microparticles from megakaryocytes: Meg-01 cells in logarithmic growth phase were grouped according to the drugs used for screening. The screened drugs included: Cyto D (0.2µM, 1µM), BMS-5 (0.2µM, 1µM), the ATPase inhibitor Blebbistatin (0.2µM, 1µM), the myosin light chain kinase inhibitor ML-7 (0.2µM, 1µM), and the ROCK kinase inhibitors Y-39983 and Y-27632 (2 / 5 / 10 µM). A negative control group (DMSO) and a positive control group (PMA, 10 ng / mL) were also included. Treatment lasted 24 hours. Cell debris was removed by two 1500 g centrifugations for 10 minutes each, yielding cell culture supernatant containing microparticles. Microparticles were analyzed by double staining with Annexin V (phosphatidylserine marker) and CD41 (megakaryocyte / platelet marker) antibodies. Microspheres were counted at 4 μm, and the level of megakaryocyte microparticles was quantitatively analyzed by flow cytometry. Step 2, verifying the role of cytoskeleton regulators in promoting megakaryocyte microparticle release: Given the reported effects of ROCK kinase inhibitors, we selected four drugs—Cyto D, BMS-5, and Blebbistatin—to further verify their role in promoting megakaryocyte microparticle release. Logarithmically growing Meg-01 cells were divided into three groups: a negative control group (DMSO), a Cyto D group (Cyto D, 0.25 / 0.5 / 1 µM, ML-7 group (0.25 / 0.5 / 1 µM), a BMS-5 group (0.25 / 0.5 / 1 µM), and a Blebbistatin group (Bleb, 0.25 / 0.5 / 1 µM), and treated for 24 hours. Flow cytometry was used to quantitatively analyze the level of megakaryocyte microparticles.

[0023] Experimental results: Flow cytometry results showed that... Figure 1 As shown, compared with the control group, different concentrations of Cyto D and BMS-5 could induce the release of microparticles from Meg-01 cells. P <0.05), 1µM Blebbistatin and 10, 10 µM Y-27632 and Y-39983 significantly promoted the release of microparticles from Meg-01 cells ( P <0.05); Flow cytometry results showed that... Figure 2 As shown, compared with the control group, Cyto D, BMS-5, ML-7, and Blebbistatin all significantly induced Meg-01 cells to release large amounts of Annexin V and CD41 double-positive microparticles. P < 0.05), with Cyto D showing the most significant effect, inducing an approximately 6-fold increase in MP release at a concentration of 0.25 µM. This result confirms that the drug of this invention exerts its effect by inducing the release of procoagulant microparticles from megakaryocytes.

[0024] Possible mechanisms by which different cytoskeleton regulators promote the release of microparticles from megakaryocytes, such as... Figure 3 As shown.

[0025] This embodiment demonstrates, using the Meg-01 cell microparticle release model and flow cytometry, that cytoskeleton regulators such as Cyto D, ML-7, BMS-5, and Blebbistatin can promote cell microparticle release by intervening in cytoskeleton regulatory pathways.

[0026] Example 2, Effects of Cytoskeleton Modulators on In Vitro Coagulation Function: To verify the direct procoagulant effect of the drugs described in this invention, this example uses Rotation Thromboelastography (ROTEM) technology to evaluate the effects of various cytoskeleton modulators on the coagulation process of fresh human whole blood, and uses classical coagulation analysis to evaluate their effects on activated partial thromboplastin time (APTT) and prothrombin time (PT).

[0027] Experimental methods: Step 1, ROTEM analysis: Fresh whole blood was collected from healthy volunteers and anticoagulated with sodium citrate. Blood samples were divided into several groups: control group (DMSO), Cyto D group (2 µM), BMS-5 (5 µM), ML-7 (2 µM), Y-39983 (5 µM), and Blebbistatin (5 µM). After incubation of each drug with the blood samples, the extrinsic coagulation pathway was initiated using EXTEM reagent, and the samples were immediately analyzed using a ROTEM delta analyzer. Parameters such as clotting time (CT), clot formation time (CFT), and maximum clot stiffness (MCF) were recorded.

[0028] The second step is to analyze APTT and PT coagulation indicators. Step 1, Particle Preparation: Fresh sodium citrate-anticoagulated whole blood was collected from healthy volunteers and platelet-rich plasma (PRP) was prepared by low-speed centrifugation (200g, 15 minutes). The PRP was aliquoted into four groups: control group (with an equal volume of DMSO), Cyto D group (2 µM), ML-7 group (2 µM), BMS-5 group (5 µM), Y-39983 group (5 µM), and Blebbistatin group (5 µM). Each group was incubated at 37°C for 1 hour to fully induce particle release. Subsequently, platelets were removed by two high-speed centrifugations (2000 g, 10 minutes), and the supernatant was carefully aspirated; this was the corresponding "particle supernatant" for each group. Step 2, APTT and PT activity assays: Take the above-mentioned sodium citrate-anticoagulated whole blood, centrifuge at 1500 g for 15 minutes at room temperature, and collect the supernatant platelet-poor plasma (PPP). Aliquot the PPP and mix it with an equal volume of each group's "microparticle supernatant". Modified APTT assay: Add APTT reagent (without activator) to the treated plasma, incubate, then add calcium chloride, and record the plasma coagulation time. PT assay: Add PT reagent (containing tissue factor) to the treated plasma, and record the plasma coagulation time.

[0029] Experimental results: ROTEM results: Compared with the blank control group, the CT and CFT values ​​of the four cytoskeleton regulators (Cyto D, BMS-5, Blebbistatin, and Y-39983) treatment groups were significantly shortened. P The value <0.05 indicates that these drugs can significantly accelerate the initiation of coagulation and the formation of blood clots. There was no significant change in maximum clot stiffness (MCF), suggesting that the drugs primarily affect the early stages of coagulation, rather than the final physical strength of the blood clot. Results are shown in Table 1 below.

[0030] Group CT (seconds) CFT (seconds) MCF (mm) DMSO 78.2 ± 5.5 91.4 ± 8.1 63.1 ± 3.8 Cyto D <![CDATA[49.5 ± 4.1 * ]]> <![CDATA[56.2 ± 6.3 * ]]> 64.0 ± 4.2 BMS-5 <![CDATA[55.1 ± 4.8 * ]]> <![CDATA[63.8 ± 7.0 * ]]> 62.5 ± 3.5 Blebbistatin <![CDATA[51.7 ± 5.0 * ]]> <![CDATA[59.9 ± 6.8 * ]]> 63.8 ± 4.0 ML-7 76.1 ± 3.2 65.9 ± 6.2 69.2 ± 7.2 * Note: Compared with the control group, P <0.05.

[0031] APTT and PT coagulation index analysis: Compared with the control group, all three cytoskeleton regulators (Cyto D, BMS-5, and Blebbistatin) significantly shortened APTT and PT in platelet-poor plasma. Cyto D showed a particularly pronounced shortening effect on APTT, consistent with the mechanism of action of the drug in this invention, which induces the release of microparticles carrying phospholipid surfaces from cells, thereby providing a key catalytic surface for coagulation factor complexes in the intrinsic coagulation pathway. The shortening of PT further confirms its accelerating effect on the common coagulation pathway. Results are shown in Table 2 below.

[0032] Group APTT(s) PT(s) DMSO 37.5 ± 2.1 17.8 ± 0.9 Cyto D <![CDATA[29.1 ± 1.8 * ]]> <![CDATA[15.2 ± 0.7 * ]]> BMS-5 <![CDATA[32.8 ± 2.0 * ]]> 16.9 ± 0.8 Blebbistatin <![CDATA[31.2 ± 1.9 * ]]> <![CDATA[14.5 ± 0.6 * ]]> ML-7 34.5 ± 5.1 16.8 ± 0.5 * Note: Compared with the control group, P <0.05.

[0033] This embodiment demonstrates, through two types of in vitro experiments—ROTEM and classical coagulation—that cytoskeleton regulators targeting various targets, such as Cyto D, BMS-5, and Blebbistatin, all possess potent and rapid direct procoagulant activity.

[0034] Example 3, In vivo hemostatic effect of cytoskeleton regulator in mouse tail tip hemorrhage model: To verify the actual hemostatic efficacy of the drug of the present invention in vivo, a classic mouse tail tip hemorrhage model was established in this example.

[0035] Experimental methods: Step 1: Cytoskeleton regulator-induced release of cellular microparticles in vivo: Male C57BL / 6 mice, 8-12 weeks old. Mice were randomly divided into 5 groups (n=4 / group): solvent control group (intravenous injection, physiological saline containing 10% DMSO and 18% sulfobutyl-β-cyclodextrin), Cyto D group (intravenous injection, 5 vs 20 mg / kg), BMS-5 group (intravenous injection, 5 vs 20 mg / kg), Blebbistatin group (intravenous injection, 2 vs 10 mg / kg), and Y-39983 (intravenous injection, 2 vs 10 μM). The injection dose was 100 μL / mouse. Peripheral blood was collected from the mandibular vein at 15, 30, 120, and 24 minutes after a single administration. Plasma was separated twice by centrifugation at 1500 g using PBS filtered through a 0.1 μm membrane at a 1:9 ratio. The number of Annexin V and CD41 double-positive cellular microparticles in the plasma was detected by flow cytometry. The second step involved using cytoskeleton regulators to accelerate hemostasis: Male CD41 knockout C57BL / 6 mice, aged 8-12 weeks, were used. Their body weight (W) was recorded before the experiment. Mice were randomly divided into four groups (n=6 / group): solvent control group, Cyto D group (intravenous injection, 2 mg / kg), BMS-5 group (intravenous injection, 5 mg / kg), Blebbistatin group (intravenous injection, 5 mg / kg), and Y-39983 group (intravenous injection, 10 μM). The injection dose was 100 μL per mouse. Fifteen minutes after a single administration, the mouse tail was surgically severed 2 mm distal to the tip. The tail was immediately immersed in 10 mL of physiological saline at 37°C. The time required for bleeding to stop (no blood flow for more than 30 seconds) was recorded (bleeding time). Meanwhile, the blood cell pellet was collected by centrifugation at 500 rpm for 10 minutes, 2 mL of red blood cell lysis buffer was added, and the cells were lysed at room temperature for 5 minutes. The absorbance OD405 at a wavelength of 405 nm was measured using an ELISA reader. The total blood loss was estimated based on the hemoglobin content: All outflowing blood was collected using the following formula, and the total blood loss (μL / g body weight) was calculated based on the weight: (OD405 × 0.46) / W × 2.2 / 2, where W is body weight (g).

[0036] Experimental results: like Figure 4 As shown, the number of plasma megakaryocytes / platelet microparticles in mice in the Cyto D, BMS-5, and Blebbistatin groups was significantly higher than that in the control group 15-30 minutes after administration. P <0.05), suggesting that cytoskeleton regulators can rapidly promote the release of cellular microparticles in vivo.

[0037] like Figure 5As shown, compared with the control group, the bleeding time of mice in the Cyto D, Blebbistatin, and Y-39983 groups was significantly shortened ( P <0.05), such as Figure 5 As shown in Figure A; the total blood loss in the Cyto D and Blebbistati groups was also significantly reduced ( P <0.05), such as Figure 5 As shown in Figure B, this result strongly demonstrates that cytoskeleton regulators have a definite and efficient hemostatic effect in live animal models.

[0038] In summary, this invention, through rigorous in vitro and in vivo experiments, has for the first time demonstrated that cytoskeleton regulators possess powerful procoagulant and hemostatic functions, and elucidated their possible mechanisms of action, providing a promising new option for the treatment of hemorrhagic diseases.

[0039] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of cytoskeleton regulators in the preparation of procoagulant drugs.

2. The application of the cytoskeleton regulator according to claim 1 in the preparation of procoagulant drugs, characterized in that: The procoagulant is a drug used to treat or prevent bleeding disorders associated with impaired coagulation function.

3. The application of the cytoskeleton regulator according to claim 2 in the preparation of procoagulant drugs, characterized in that: The bleeding disorders mentioned include traumatic bleeding, surgical bleeding, liver disease-related bleeding, postpartum hemorrhage, hemophilia, bleeding caused by anticoagulant overdose, and bleeding related to thrombocytopenia or functional disorders.

4. The application of the cytoskeleton regulator according to claim 1 in the preparation of procoagulant drugs, characterized in that: The cytoskeleton regulator is a small molecule compound that targets and regulates cytoskeleton signaling pathways.

5. The application of the cytoskeleton regulator according to claim 1 in the preparation of procoagulant drugs, characterized in that: The cytoskeleton regulator is one or more of the following: compounds that inhibit actin microfilament polymerization, compounds that inhibit myosin contraction, and ROCK kinase inhibitors.

6. The application of the cytoskeleton regulator according to claim 5 in the preparation of procoagulant drugs, characterized in that: The compound that inhibits actin microfilament polymerization is cytochalasin D and / or the LIMK1 / 2 inhibitor BMS-5.

7. The application of the cytoskeleton regulator according to claim 5 in the preparation of procoagulant drugs, characterized in that: The compounds that inhibit myosin contraction are the ATPase inhibitor Blebbistatin and / or the myosin light chain kinase inhibitor ML-7.

8. The application of the cytoskeleton regulator according to claim 5 in the preparation of procoagulant drugs, characterized in that: The ROCK kinase inhibitor is Y-39983 and / or Y-27632.

9. The application of the cytoskeleton regulator according to claim 5 in the preparation of procoagulant drugs, characterized in that: The procoagulant includes cytoskeleton regulators and pharmaceutically acceptable excipients.

10. The application of the cytoskeleton regulator according to claim 5 in the preparation of procoagulant drugs, characterized in that: The excipients are one or more of the following: fillers, stabilizers, diluents, adjuvants, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.

Images