Application of harmine in preparation of thrombolytic drug

Abstract

The invention discloses a novel application of harmine in preparation of thrombolytic drugs. The research shows that harmine can effectively dissolve formed thrombus and relieve vasculopathy, and has the function of protecting blood vessels. After thrombus is formed, harmine is given, and thrombolysis treatment can be achieved through the following steps that the blood flow speed and the blood flow volume of the thrombus part are rapidly recovered; platelet activation and endothelial cell injury are obviously inhibited; and oxidative stress is eliminated. The blood flow disorder caused by thrombus can be quickly improved; formed thrombus is effectively dissolved; the normal vasomotor function is maintained; the physiological hemostasis function is not interfered. The harmine is remarkable in thrombolysis effect and quick in action; the curative effect is lasting; the safety is high and the bleeding risk is low. The invention provides a brand new drug choice for treating thrombotic diseases, and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and more particularly to the application of harmine in the preparation of thrombolytic drugs. Background Art

[0002] Thrombotic diseases (such as myocardial infarction, stroke, and deep vein thrombosis) are common clinical critical illnesses. While existing thrombolytic drugs (such as t-PA and urokinase) can dissolve clots by activating the fibrinolytic system, they still have the following limitations: a strict time window (must be used within 4.5 hours of clot formation; efficacy is significantly reduced after this window); a high risk of bleeding (systemic fibrinolytic activation may lead to intracranial hemorrhage (incidence 1%-2%)); vascular reocclusion (10%-25% of patients experience recurrent thrombus formation due to endothelial damage and platelet reactivation after thrombolysis); and ineffectiveness in stabilizing clots (difficulty dissolving old clots with high fibrosis). Current clinically available thrombolytic drugs target only fibrinolysis and lack multiple mechanisms of action, including endothelial protection, platelet activation regulation, and improvement of the thrombotic microenvironment. Although newer antiplatelet drugs (such as ticagrelor) and anticoagulants (such as rivaroxaban) can reduce the risk of thrombosis, they have no effect on existing clots. Therefore, it is urgent to develop multifunctional thrombolytic agents that combine rapid thrombolysis, vascular protection and microenvironment regulation.

[0003] Harmine (HAR) is a natural dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor with anticancer and anti-inflammatory activities. Patent CN119792282A discloses the use of harmine in the preparation of antiviral drugs; Patent CN117717547A discloses the use of harmine in the treatment of CCNE1-amplified ovarian cancer; and Patent CN116251098A discloses the use of harmine in the preparation of drugs for the treatment and / or prevention of pulmonary arterial hypertension. However, there are currently no reports on the effects of harmine on established thrombi. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned defects and deficiencies in the prior art and to provide the use of harmine in the preparation of thrombolytic drugs. The present invention demonstrates that harmine has a novel application in the preparation of thrombolytic drugs by dissolving established thrombi, protecting vascular function, and regulating multiple targets.

[0005] The second object of the present invention is to provide the use of harmine in the preparation of medicines for treating thrombotic diseases.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions: This invention reveals for the first time that dehydrogenated echinopsine achieves efficient thrombolytic therapy through a multi-target synergistic mechanism, specifically including: ① Rapid thrombolytic effect: within 1 hour after administration after thrombosis, hemodynamic recovery is achieved, and the peak systolic velocity (PSV) increases from 55.80±6.92 cm / s to 254.51±29.84 cm / s (recovery rate 75.8%). # P<0.05 vs Model), thrombus dissolution rate 61.2% (histopathological quantification, # P<0.05 vs Model);② Vascular protection: Improved vascular elasticity, and the blood flow time integral (VTI) recovered from 6.46±2.36 to 22.76±7.07 (recovery rate 82.3%, # P<0.05 vs Model); maintain coagulation homeostasis: pulsatility index (PI) 1.03±0.12 (no statistical difference compared with the control group 1.14±0.06, *P>0.05 vs Control); ③ Multi-target regulation of thrombotic microenvironment: inhibit platelet activation, and the number of P-selectin-positive platelets decreased by 67.5% ( ## P<0.01 vs Model), the expression intensity (MFI) decreased by 45.1% ( ## P<0.01 vs Model); inhibition rate of vascular endothelial P-selectin expression was 54.6% ( ## P<0.01 vs Model); clearing oxidative stress, the local ROS fluorescence intensity of thrombus decreased by 84.8% ( ## P < 0.01 vs Model).

[0007] Therefore, the present invention first provides the use of harmine in the preparation of thrombolytic drugs. Harine achieves a new use in the preparation of thrombolytic drugs by effectively dissolving formed thrombi, protecting vascular function, and regulating the thrombus microenvironment at multiple targets.

[0008] The invention further provides the use of dehydrogenated harmine in preparing a multifunctional thrombolytic agent with the functions of rapid thrombolysis, vascular protection and improvement of thrombus microenvironment.

[0009] The present invention also provides the use of dehydrogenated harmine in preparing a medicine for treating thrombotic diseases requiring both thrombolytic efficiency and safety.

[0010] The term "treating" refers to achieving treatment, alleviation and / or relief of the diseases or conditions described herein in a subject.

[0011] Furthermore, the thrombotic disease includes but is not limited to acute arterial thrombotic disease, venous thromboembolism, diabetes-related thrombosis, microvascular thrombosis-related disease or iatrogenic thrombosis, etc. The acute arterial thrombosis includes stroke, myocardial infarction, etc.

[0012] Specifically, the drug achieves treatment by rapidly restoring blood flow velocity and blood volume at the thrombus site, improving vascular elasticity, maintaining coagulation homeostasis, inhibiting platelet activation and endothelial inflammation, and clearing oxidative stress.

[0013] The drug can be used in patients or other animals that receive the drug of the present invention to treat, alleviate and / or relieve the diseases or conditions described in the present invention. In a preferred embodiment, the drug is used in mammals, including but not limited to humans, cattle, horses, sheep, pigs, goats, rabbits, cats, dogs, mice, and any other mammals that can produce thrombosis.

[0014] The dosage of the drugs of the present invention depends on many factors, such as the nature and severity of the condition being treated, the sex, age, weight, and individual response of the patient or animal, the route of administration, and the number of doses. The dosage may be administered as a single dose or divided into several doses, such as two, three, or four. The dosage level should be selected based on the specific route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated.

[0015] However, it should be understood that the total daily dosage of the drugs of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate; the duration of treatment; any combination or concurrent medications; and similar factors well known in the medical field. For example, it is common practice in the art to start administration at a dose lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0016] Furthermore, the dosage of harmine is 5 to 15 mg / kg.

[0017] Preferably, the dosage of harmine is 10 mg / kg.

[0018] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage form of the drug includes but is not limited to injection or oral preparation.

[0020] The present invention discovered and confirmed for the first time that dehydrogenated eugeniacin has the effect of dissolving formed thrombi, clarified that it exerts thrombolytic effects through the synergistic action of multiple targets, and developed a new safe and efficient thrombolytic therapy regimen. These innovations make the present invention significantly superior to existing technologies in terms of thrombolytic therapy effect, safety and scope of application, and it has important clinical application value and broad market prospects.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a new use of harmine in the preparation of thrombolytic drugs. Harmine can effectively dissolve formed thrombi and alleviate vascular lesions, with both thrombolytic and vascular protective effects. It has a clinical breakthrough effect: rapid thrombolysis: PSV recovery ≥ 75% within 1 hour of administration ( # P<0.05 vs Model), thrombus dissolution rate 61.2% ( # P<0.05 vs Model); Precision safety: Maintained normal PI value (*P>0.05 vs Control), no bleeding or coagulation dysfunction was observed; Multi-mechanism synergistic protection: Vascular function maintenance: VTI recovered 82.3% ( # P<0.05 vs Model), resistance index (RI) improved from 0.53±0.05 to 0.63±0.03 ( # P<0.05 vs Model), microenvironment regulation: simultaneous inhibition of platelet activation (P-selectin↓67.5%, ## P<0.01 vs Model), vascular endothelial P-selectin expression inhibition rate 54.6% ( ## P<0.01 vsModel) and the ROS fluorescence intensity in the thrombus was reduced by 84.8% ( ## P<0.01 vs Model). Expanded clinical indications: Suitable for thrombotic diseases requiring both thrombolytic efficiency and safety, such as acute arterial thrombosis (e.g., stroke, myocardial infarction), venous thromboembolism, and diabetes-related thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of dehydrogenated echinopsine on FeCl3-induced carotid artery thrombus burden in mice.

[0023] Figure 2 To investigate the effect of harmine on the pathological structure and functional recovery of vascular tissue after thrombus dissolution.

[0024] Figure 3 This is the improvement effect of dehydrogenated echinopsine on the hemodynamic parameters of the thrombosis model (evaluated by high-frequency ultrasound).

[0025] Figure 4 Harmine selectively clears thrombosis-induced endothelial ROS (DCFH-DA fluorescence detection).

[0026] Figure 5 Harmine specifically inhibits thrombosis-induced endothelial P-selectin expression.

[0027] Figure 6 Harmine blocks the activation cascade by inhibiting platelet P-selectin expression (flow cytometry analysis). Specific Embodiments

[0028] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Harmine is a product of MedChemExpress, with the product catalog number HY-N0737A, purity of 99.88%, CAS No.: 442-51-3. Its structure is as follows: .

[0031] C57 / 6J mice are all products of Guangdong Medicilon Experimental Animal Technology Co., Ltd., SYXK (Yue) 2022-0079.

[0032] Statistical analysis: In the following embodiments, the experimental data are expressed as mean ± standard deviation (Mean ± SD), n = 6. The t-test is used for comparison between groups, and *P < 0.05 indicates significant difference.

[0033] Example 1 Establishment of an animal model of arterial thrombosis and drug evaluation method 1. Method (1) Experimental animals and grouping Take 8-week-old male C57BL / 6J mice (purchased from Guangdong Medicilon Experimental Animal Technology Co., Ltd., license number SYXK (Yue) 2022-0079), and randomly divide them into the following 4 groups (n = 6): Control group: sham operation (no induced thrombosis) + intraperitoneal injection of normal saline; Harmine group: sham operation (no induced thrombosis) + intraperitoneal injection of harmine (10 mg / kg); Model group: FeCl3-induced thrombosis + intraperitoneal injection of normal saline; Harmine + Model group: FeCl3-induced thrombosis + intraperitoneal injection of harmine (10 mg / kg).

[0034] (2) Establishment of the thrombosis model After anesthetizing and fixing the mice, the neck was depilated and disinfected. A longitudinal incision was made along the anterior midline of the neck, with the length adjusted to fully expose the surgical field. The common carotid artery was exposed by blunt dissection to ensure the integrity of the vascular adventitia. A 5% FeCl3 solution-induced material was applied to the outer wall of the blood vessel for 3 minutes. After the induction material was removed, the blood vessel was rinsed with normal saline. After 30 minutes of modeling, thrombosis was verified using a high-frequency ultrasound system (16-21 MHz) (PSV < 100 cm / s). The corresponding drugs were immediately injected intraperitoneally, and various indicators were measured 1 hour after treatment.

[0035] (3) Evaluation indicators and detection methods Ultrasound hemodynamic testing: A high-frequency ultrasound system (probe frequency 16-21 MHz) measures parameters including peak systolic velocity (PSV), end-diastolic velocity (EDV), time-averaged velocity (TAMV), the resistance index (RI) = (PSV-EDV) / PSV, and the pulsatility index (PI) = (PSV-EDV) / TAMV. PSV and EDV reflect blood flow velocity characteristics, while the pulsatility index (VTI) indicates blood flow during the cardiac cycle. The RI is used to assess the degree of vascular stenosis and distal resistance.

[0036] Quantitative analysis of thrombus: A 6-8 mm thrombus segment was weighed and the thrombus burden (thrombus mass / length ratio (mg / cm)) was calculated.

[0037] Histopathological evaluation: Vascular samples were fixed with 4% paraformaldehyde for 6-12 hours and routinely embedded in paraffin for 3-5 μm sections. Conventional H&E staining (hematoxylin for 2 minutes / eosin for 30 seconds) was used to observe thrombus morphology. The thrombus lysis rate was quantitatively calculated under an optical microscope as (model group area - harmine group area + model group area) / model group area × 100%.

[0038] 2. Results (1) Thrombolytic effect of harmaline and improvement of hemodynamics After treatment with dehydrogenated echinopsine, the thrombolytic effect and hemodynamic parameters of thrombosis model animals were significantly improved (Table 1, Figure 1 AH). Specifically, after drug administration, the peak flow velocity (PSV) recovered to about 75.8% of the control group level (254.51 / 335.78 cm / s, # P<0.05vs Model), blood flow time integral (VTI) recovered to about 82.3% (22.76 / 28.25, # P<0.05 vs Model), resistance index (RI) recovered from 0.53±0.05 to 0.63±0.03 ( #P<0.05 vs. Model. The pulsatility index (PI) remained at 1.03±0.12 (not statistically different from 1.14±0.06 in the control group, *P>0.05), indicating that the drug did not affect baseline vascular function. The degree of recovery in PSV and VTI, along with the changes in RI and PI, suggests that harmine primarily acts through a thrombolytic mechanism, with no significant vasodilatory effect observed. These results demonstrate that harmine achieves its effects through thrombolysis (not vasodilatory effects) with high efficacy (PSV recovery ≥75%), targeted efficacy (significant improvement in RI), and safety (stable PI).

[0039] Table 1 Effects of harmine on hemodynamics in thrombosis model (Mean±SD, n=6)

[0040] Note: ** P < 0.01 vs Control; # P < 0.05 vs Model; all unmarked values ​​are P > 0.05; recovery rate = (value of the Harmine + Model group / value of the Control group) × 100%.

[0041] (2) Dissolution of FeCl3-induced thrombus formation by dehydrogenated harmine A FeCl3-induced carotid artery thrombosis model was established in mice. High-frequency ultrasound (Vevo 2100) confirmed blood flow occlusion (PSV < 100 cm / s) 30 minutes after modeling. Thrombus burden (thrombus weight / vessel length) was measured 1 hour after intervention with dehydrogenated harmine (10 mg / kg). Figure 2 As shown in Table 2, the thrombus burden in the Model group was significantly higher than that in the Control group (2.38±1.04 vs 1.04±0.33 mg / cm, * P<0.05); in the Harmine+Model group, the thrombus burden was reduced by 50.8% compared with the Model group (1.17±0.45 mg / cm, # P<0.05), and the weight of thrombus decreased simultaneously (0.62±0.21 vs 1.14±0.48 mg, # P<0.05). The above data indicate that harmine can effectively dissolve formed thrombi and alleviate vascular lesions.

[0042] Table 2 Thrombolytic effect of harmine (Mean±SD, n=6)

[0043] Note: *P < 0.05 vs Control; # P < 0.05 vs Model; *** P < 0.001 vs Control; ## P < 0.01 vs Model; all unmarked values ​​are P > 0.05.

[0044] (3) Harmine improves vascular structure and function after thrombus dissolution Vascular pathology improved after treatment with harmine as shown in Table 2 and Figure 3 As shown. The Model group showed thrombus attachment and inflammatory infiltration in the blood vessels, while the above lesions were significantly alleviated in the Harmine + Model group ( Figure 3 A). Figure 3 B shows that the thrombus area accounts for about 78.34% *** (Model group) dropped to 30.41% ## (Harmine + Model group), the dissolution rate was approximately 61.2% (#P < 0.05). These results suggest that harmine has both thrombolytic and vascular protective effects.

[0045] Example 2 Inhibitory effect of harmine on the mean fluorescence intensity of thrombotic ROS Based on the establishment of the arterial thrombosis animal model and the dosing regimen in Example 1, the effect of harmine on the thrombotic microenvironment was evaluated.

[0046] 1. Method ROS were detected using the DCFH-DA probe at a working concentration of 100 μM.

[0047] Tissue pretreatment: The thrombus segment was cut longitudinally, incubated in 3% H2O2 solution in the dark for 10 minutes (to inactivate endogenous peroxidase), and rinsed with PBS three times (2 minutes each time).

[0048] Fixation and permeabilization: Fix with 4% paraformaldehyde (PFA) for 20 minutes (room temperature, protected from light), and rinse three times with PBS.

[0049] Probe incubation: Add DCFH-DA working solution (100 μM), incubate in the dark for 1 hour (room temperature), and rinse three times with PBS.

[0050] Mounting and imaging: Anti-fluorescence fading mounting medium containing DAPI (10 μL), laser confocal microscope system, excitation / emission wavelength: 488 nm / 525 nm (Alexa Fluor 488 channel).

[0051] Data analysis: ImageJ software was used to analyze the mean fluorescence intensity (MFI), and the inhibition rate was calculated as (Model group - treatment group) / (Model group - Control group) × 100%.

[0052] 2. Results The results of DCFH-DA fluorescent probe detection of thrombotic ROS are shown in Figure 4 and Table 3. Figure 4 A shows the distribution characteristics of ROS in the thrombus site of each group. The statistical results are shown in Figure 4 B. Quantitative detection and statistical results (Table 3) showed that ROS levels in the Model group increased significantly to 43.91±17.58 (***P<0.001 vs. 8.45±4.62 in the Control group), decreased to 13.83±4.57 in the Harmine+Model group (inhibition rate 84.8%, ##P<0.01 vs. Model), and 8.92±3.84 in the Harmine group (P>0.05 vs. Control). This indicates that harmine significantly inhibits thrombosis-related ROS levels (inhibition rate ≥65%, ##P<0.01) without affecting physiological ROS levels (P>0.05).

[0053] Table 3 Inhibitory effect of harmine on the mean fluorescence intensity of thrombotic ROS (Mean±SD, n=6)

[0054] Note: *** P < 0.001 vs Control; ## P < 0.01 vs Model; all unmarked values ​​are P > 0.05.

[0055] Example 3 Harmine inhibits vascular endothelial P-selectin expression 1. Method Sample pretreatment: The thrombus segment of the blood vessel was obtained and cut longitudinally (according to the standard vascular dissection method in the field), incubated in 3% H2O2 solution in the dark for 10 minutes (to inactivate endogenous peroxidase), and rinsed with PBS three times (2 minutes each time).

[0056] Fixation and blocking: Fix with 4% paraformaldehyde (PFA) for 20 minutes (room temperature, protected from light), and block with 100 μL of 1-5% BSA blocking solution for 1 hour.

[0057] Antibody incubation: Primary antibody P-selectin mouse anti-1:500 incubation at 4℃ for 12-16 hours, corresponding species secondary antibody 1:200 incubation in the dark at room temperature for 1 hour.

[0058] Confocal microscopy detection: P-selectin: Alexa Fluor 594 (excitation 561 nm), nuclear staining: DAPI.

[0059] Quantitative analysis: ImageJ software was used to analyze the mean fluorescence intensity (MFI), and the inhibition rate was calculated as (Model group - treatment group) / (Model group - Control group) × 100%.

[0060] 2. Results Figure 5 A shows the expression distribution of P-selectin in vascular endothelium. The statistical results are shown in Figure 5 B, quantitative detection results are shown in Table 4, P-selectin mean fluorescence intensity (MFI): Model group 29.57±6.83 ( *** P<0.001 vs Control group 12.76±4.65), Harmine+Model group 20.46±6.07 (inhibition rate 54.6%, ## P<0.01 vs Model), Harmine group: 13.25±5.12 (P>0.05 vs Control). This shows that dehydrogenated harmine can significantly inhibit the expression of P-selectin in vascular endothelium induced by thrombosis (inhibition rate ≥50%). ## P < 0.01), and did not interfere with the basal expression level (P > 0.05).

[0061] Table 4 Results of endothelial P-selectin MFI detection (Mean±SD, n=6)

[0062] Note: ***P<0.001 vs Control; ## P < 0.01 vs Model; all unmarked values ​​are P > 0.05.

[0063] Example 4 Harmine inhibits platelet P-selectin expression 1. Method Flow cytometry was used to assess platelet P-selectin expression: 20 μL of fresh blood was collected from each group and lysed with 100 μL of red blood cell lysis buffer for 10 minutes. Lysis was terminated with 1 mL of PBS, centrifuged at 800 g for 10 minutes, and the supernatant removed. The cells were washed with 1 mL of PBS, mixed, and centrifuged at 800 g for 10 minutes, and the supernatant removed. The cells were incubated with 100 μL of PBS containing CD62P-PE antibody for 30 minutes, followed by 1 mL of PBS. The platelets were then assayed using a flow cytometer. The inhibition rate was calculated as (Model group - Harmine + Model group) / (Model group - Control group) × 100%.

[0064] 2. Results The expression of P-selectin in mouse platelets was detected by flow cytometry. The results are shown in Table 5 and Figure 6 As shown in AB. All parameters in the Model group were significantly increased ( *** P < 0.001 vs Control: P-selectin-positive platelet count: 3426 ± 1162 (Control: 1276 ± 791), activation ratio: 62.4 ± 22.7% (Control: 25.5 ± 15.8%), MFI: 2847 ± 673 (Control: 1024 ± 258). Harmine inhibited the P-selectin-positive platelet count: 1974 ± 509 (inhibition rate: 67.5%, ##P < 0.01 vs Model), activation ratio: 42.3 ± 10.2% (inhibition rate: 32.2%, ##P < 0.01), MFI: 1562 ± 421 (inhibition rate: 45.1%, ##P < 0.01). The results showed that harmine reduced the number of activated platelets (inhibition rate ≥ 65%), reduced the overall activation level (inhibition rate ≥ 30%), and attenuated the intensity of P-selectin expression (inhibition rate ≥ 45%).

[0065] Table 5 Regulatory effect of harmine on platelet P-selectin expression (Mean±SD, n=6)

[0066] Note: *** P < 0.001 vs Control; ## P < 0.01 vs Model; all unmarked values ​​are P > 0.05.

Claims

1. Application of dehydrogenated harmine in the preparation of thrombolytic drugs.

2. Application of dehydrogenated echinopsine in the preparation of a multifunctional thrombolytic agent with rapid thrombolysis, vascular protection and improvement of thrombotic microenvironment.

3. Application of dehydrogenated harmine in the preparation of drugs for treating thrombotic diseases.

4. The application according to claim 3, characterized in that The thrombotic disease is selected from acute arterial thrombotic disease, venous thromboembolism, diabetes-related thrombosis, microvascular thrombosis-related disease or iatrogenic thrombosis.

5. The use according to any one of claims 1 to 4, characterized in that: The drug achieves treatment by rapidly restoring the blood flow velocity and blood volume at the thrombus site, improving vascular elasticity, maintaining coagulation homeostasis, inhibiting platelet activation and endothelial inflammation, and clearing oxidative stress.

6. The use according to any one of claims 1 to 4, characterized in that: The dosage of the dehydrogenated harmine is 5 to 15 mg / kg.

7. The application according to claim 6, characterized in that The administration dosage of harmine is 10 mg / kg.

8. The use according to any one of claims 1 to 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the medicine is injection or oral preparation.

10. The use according to claim 9, characterized in that: The dosage form of the medicine is injection.

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