Application of pantethine in preparation of medicine for preventing and / or treating thrombotic diseases

Abstract

The invention belongs to the technical field of medicines, and discloses application of pantethine in preparation of medicines for preventing and / or treating thrombotic diseases or medicines for inhibiting platelet activation or regulating vascular endothelial cell functions. In-vivo and in-vitro experiments prove that pantethine can remarkably relieve thrombus load in a mouse thrombus model, down-regulate expression of vWF (von Willebrand Factor), up-regulate expression of TFPI (Tissue Factor Path Inhibitor) and TH BD (Thromboregulatory Protein), and antagonize platelet adhesion, aggregation and activation induced by a platelet activator. The action mechanism is closely related to inhibition of reactive oxygen species (ROS) generation and NF-kappa B signal channel activation, and improvement of endothelial cell functions. It can be known from the invention that pantethine plays an antithrombotic role from multiple aspects, and is suitable for preparation of antithrombotic drugs for prevention or treatment. The invention provides a new drug choice for prevention and treatment of thrombotic diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to application of pantethine in preparation of a medicine for preventing and / or treating thrombotic diseases. BACKGROUND

[0002] Thrombotic diseases, such as deep vein thrombosis, pulmonary embolism, arterial thrombosis, etc., are one of the main causes of cardiovascular events and death. Currently, the commonly used antithrombotic drugs in clinical practice mainly include antiplatelet drugs (such as aspirin, clopidogrel) and anticoagulants (such as rivaroxaban, warfarin, heparin) and the like. Although these drugs play an important role in the prevention and treatment of thrombosis, there are still problems such as high risk of bleeding, large individual differences, many drug interactions, drug resistance in some patients, etc.

[0003] Recent studies have shown that oxidative stress is closely related to endothelial dysfunction, excessive activation of platelets and disorders of the coagulation system, and is an important inducer of thrombosis. Accumulation of reactive oxygen species (ROS) can activate inflammatory signaling pathways such as NF-κB, promote the expression of pro-coagulation / adhesion factors such as von Willebrand factor (vWF) and intercellular adhesion molecule-1 (ICAM-1), and inhibit the expression of anticoagulant proteins such as thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI), thereby leading to a prothrombotic state.

[0004] Pantethine is a derivative of pantothenic acid (vitamin B5), its chemical name is D-bis (N-pantothenyl-beta-aminoethyl) disulfide, its molecular formula is C 22 H 42 N4O8S2, and its molecular weight is 554.72, and its specific structural formula is as follows:

[0005]

[0006] As a precursor of coenzyme A (CoA) and acyl carrier protein (ACP), it plays an important role in fatty acid metabolism, tricarboxylic acid cycle and antioxidant defense. Pantethine is an important precursor of coenzyme A biosynthesis, which can be converted to 4'-phosphopantetheine in vivo, and then participate in various key biochemical processes such as fatty acid metabolism and tricarboxylic acid cycle. The pantethine described in the present application can be prepared by chemical synthesis or microbial fermentation, and the purity can reach more than 98%, which meets the pharmaceutical standards.

[0007] Recent studies have found that pantethine has pharmacological activities of improving lipid metabolism and reducing lipids, but its role in antithrombosis has not been systematically studied, and there is no report of its use in the prevention or treatment of thrombotic diseases. Through relevant experimental research, the present application confirms the possibility of pantethine as a drug for preventing and treating thrombotic diseases from multiple aspects. SUMMARY

[0008] The application aims to provide a use of pantethine in the preparation of a drug for preventing and / or treating thrombotic diseases or inhibiting platelet activation or regulating vascular endothelial cell function.

[0009] The application provides a use of pantethine in the preparation of a drug for preventing and / or treating thrombotic diseases or inhibiting platelet activation or regulating vascular endothelial cell function.

[0010] The pantethine comprises a pharmaceutically acceptable salt, a solvate or a prodrug thereof.

[0011] The drug is a single preparation or a compound preparation.

[0012] The thrombotic diseases include, but are not limited to, deep vein thrombosis, pulmonary embolism, arterial thrombosis, cerebral thrombosis or disseminated intravascular coagulation.

[0013] The drug can be a single preparation or a compound preparation, and the dosage form includes, but is not limited to, a tablet, a capsule, an oral liquid, an injection, a freeze-dried powder injection, a sustained-release preparation and the like.

[0014] The pantethine plays an anti-thrombotic role by inhibiting the generation of active oxygen and the activation of an NF-κB signal pathway.

[0015] The pantethine regulates the blood coagulation balance by down-regulating the expression of a von Willebrand factor vWF and up-regulating the expression of a tissue factor pathway inhibitor TFPI and / or a thrombomodulin THBD, and preferably, the pantethine plays an anti-thrombotic role by inhibiting platelet adhesion, aggregation and / or activation.

[0016] The drug further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0017] The application provides a pharmaceutical composition comprising a therapeutically effective amount of pantethine and one or more pharmaceutically acceptable carriers.

[0018] Preferably, the daily dose of the pantethine per kilogram of mice is 250 mg to 750 mg, the daily dose of the pantethine for an adult is 100 mg to 600 mg, and the mass concentration of the pantethine in the pharmaceutical composition accounts for at least 20%.

[0019] Based on in-depth in-vivo and in-vitro experimental research, the application first systematically clarifies a new use of pantethine in synergistically playing an anti-thrombotic role through multiple targets and multiple pathways.

[0020] Mechanism of the application: Pantethine as a pantothenate derivative in the application plays an antithrombotic role by regulating endothelial cell function, inhibiting platelet activation and regulating the expression of coagulation-related factors. Experimental results show that pantethine significantly reduces thrombus load in a mouse thrombus model, inhibits arachidonic acid (AA)-induced platelet activation and aggregation, down-regulates pro-coagulation factors vWF and ICAM1 expression, and up-regulates anti-coagulation factors TFPI and THBD expression. Mechanism research shows that pantethine improves endothelial cell function by inhibiting reactive oxygen species (ROS) production and NF-κB signaling pathway activation, thereby maintaining coagulation-anticoagulation balance. Pantethine is highly safe and suitable for the preparation of a drug for preventing or treating thrombotic diseases.

[0021] Advantages: Compared with the prior art, the application has the following outstanding advantages: the application first systematically reveals the antithrombotic effect and mechanism of pantethine; has multi-target action characteristics, covering multiple links such as platelets, endothelial cells, and coagulation systems; has high safety, a wide treatment window, and no obvious toxic side effects in long-term use; and specifically has the following functions:

[0022] 1. Overall improvement of endothelial cell function: pantethine can reverse the pro-coagulation phenotype of endothelial cells induced by inflammatory factors, specifically down-regulate the expression of pro-coagulation / adhesion factors such as vWF and ICAM-1, and up-regulate the expression of anti-coagulation / vascular protection factors such as TFPI and THBD, thereby promoting the transformation of endothelial cells from a pro-coagulation phenotype to an anti-coagulation phenotype;

[0023] 2. Significant inhibition of platelet adhesion and aggregation induced by platelet activator arachidonic acid (AA), and significant reduction of the expression of platelet surface activation marker P-selectin;

[0024] 3. Multi-pathway regulation molecular mechanism: the antithrombotic effect of pantethine involves the synergistic regulation of multiple signaling pathways: significantly inhibits the activation of the NF-κB signaling pathway of endothelial cells, reduces the phosphorylation levels of IκB and p65, significantly reduces the ROS level of endothelial cells, and alleviates oxidative stress damage;

[0025] 4. In animal models, pantethine is found to comprehensively improve endothelial cell function, down-regulate the expression of pro-coagulation / adhesion factors such as vWF and ICAM-1, and up-regulate the expression of anti-coagulation / vascular protection factors such as TFPI, through omics analysis;

[0026] 5. Significant reduction of thrombus load in mouse inferior vena cava ligation and pulmonary embolism models: in various thrombus animal models (including inferior vena cava ligation models and pulmonary embolism models), pantethine significantly reduces thrombus load;

[0027] 6. Good safety features: no obvious toxic side effects are found in 28-day long-term animal tests, the treatment window is wide, and the safety is good. Attached Figure Description

[0028] Figure 1 The effect of panthioethylamine on the expression of vWF, THBD and TFPI mRNA in vascular endothelial cells induced by the inflammatory factor IL-1β.

[0029] Figure 2 The effect of panthioethylamine on IL-1β-induced activation of the NF-κB pathway in vascular endothelial cells.

[0030] Figure 3 The effect of panthioethylamine on ROS production in vascular endothelial cells induced by the inflammatory factor IL-1β.

[0031] Figure 4 The effect of panthioethylamine pretreatment on platelet adhesion in endothelial cells.

[0032] Figure 5 The effect of endothelial cells pretreated with panthioethylamine after stimulation with different concentrations of arachidonic acid on platelet activation.

[0033] Figure 6 The effect of arachidonic acid stimulation followed by pretreatment with panthioethylamine on platelet aggregation in endothelial cells.

[0034] Figure 7 The changes in the expression of vWF, ICAM1 and TFPI proteins in the serum of mice treated with panthioethylamine were investigated.

[0035] Figure 8 The effect of platelets isolated from mice treated with panthioethylamine on platelet activation and aggregation induced by the platelet activator arachidonic acid.

[0036] Figure 9 The effect of panthioethylamine on thrombosis in a mouse inferior vena cava (IVC) ligation model.

[0037] Figure 10 The effect of panthioethylamine on thrombus formation in a mouse pulmonary embolism model.

[0038] Figure 11 Panthionylamine possesses good safety characteristics. Detailed Implementation

[0039] Experimental materials and instruments:

[0040] Experimental materials: Pantothenic acid (purity > 99.9%, MCE, HY-B1028); human umbilical vein endothelial cell line EA.Hy926 (Zhong Qiao Xinzhou, catalog number ZQ0079); arachidonic acid (AA) (Aladdin, A131025|506-32-1), IL-1β (Abixin, catalog number abs06058), fibrinogen (MCE, catalog number HY-NP166), collagen / adrenaline (Helena Laboratories, catalog number 5369). Antibodies: Rabbit anti-NF-κB (Yuantai Bio, catalog number P20661), rabbit anti-phospho-NF-κB (Yuantai Bio, catalog number P20282), rabbit anti-phospho-IκB (MCE, catalog number HY-P80466), rabbit anti-IκB (Huayuan Bio, catalog number HY-P80466), rabbit anti-β-actin (Abmart, catalog number P30002S), goat anti-rabbit-HRP (abcam, catalog number ab6721). PerCP / Cyanine 5.5-tagged anti-CD41 (BioLegend, catalog number 303720), PE-conjugated anti-CD62P (BioLegend, catalog number 304906), PerCP / Cyanine 5.5-tagged anti-CD41 (BioLegend, catalog number 133918), PE-conjugated anti-CD62P (BioLegend, catalog number 148305). BCA kit (Beyotime, catalog number P0012S), PrimeScript RT Master Mix Perfect Real Time reverse transcription (TaKaRa, catalog number DRR0036A), Faststart Universal SYBR Green Master quantitative RT-PCR reagent (Roche, catalog number 04913914001), protease inhibitor (Roche, catalog number 11873580001), PVDF membrane (Roche, catalog number 03010040001), WB Immobilon ECL chemiluminescence solution (Millipore, catalog number WBKLS0500), DCFH-DA reactive oxygen species (ROS) fluorescent probe (Solepro, catalog number D6470-25mg), ready-to-use tribromoethanol solution / Aphrodine / 1.25% (ready-to-use anesthetic for animal experiments) (Aibei Biotechnology, catalog number M2910).

[0041] Experimental instruments: Flow cytometer (BD Biosciences), platelet aggregator (Chrono-Log), chemiluminescence imaging system (Bio-Rad), laser confocal microscope (Leica TSC SP8 X), PCR instrument (PCR Thermal CyclerDice, TaKaRa), nucleic acid purity and concentration analyzer (Biophotometer plus, eppendorf), real-time quantitative PCR instrument and analysis software (ABI7500), vertical electrophoresis tank (BIO-RAD), electrophoresis apparatus (BIO-RAD), semi-dry transfer apparatus (BIO-RAD), nano-level liquid chromatography-tandem mass spectrometry (Evosep One combined with Orbitrap Exploris 480 mass spectrometer), C18 column (75μm x 25cm, Thermo, USA).

[0042] Mouse source: Male C57BL / 6J mice, weighing 18–22 g, 2–3 months old, purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015).

[0043] In the embodiments of this invention, 750 mg / kg / day refers to a daily dose of 750 mg per kg of mouse.

[0044] Example 1: Pantothecin regulates endothelial cell coagulation-related factors by inhibiting the ROS and NF-κB pathways.

[0045] Before treatment, EA.Hy926 cells were inoculated at a concentration of 2 × 10⁻⁶. 5Cells were seeded in 6-well plates at a concentration of 100 μM / ml. When confluence reached 80%-90%, the cells were pretreated with 100 μM tebuconazole for 12 hours (with an equal volume of 0.01 M PBS at pH 7.2-7.4 as a control), followed by IL-1β treatment (50 ng / ml) for 30 minutes. After treatment, the cells were washed once with PBS, and 0.5 ml of TRIZOL was added to each well. After standing for 2 minutes, the cells were repeatedly lysed by pipetting, and the lysate was aspirated into a 1.5 ml RNase-free EP tube. 100 μl of chloroform was added, and the cells were vigorously shaken for 15 seconds, allowed to stand for 5 minutes, and then centrifuged at 12000 g, 4°C for 15 minutes. The supernatant was carefully transferred to a new RNase-free EP tube, and an equal volume of isopropanol was added. After standing for 10 minutes, the cells were centrifuged at 12000 g, 4°C for 10 minutes. Discard the supernatant, add 1 ml of pre-cooled 75% ethanol to wash the tube wall, centrifuge at 7500g, 4°C for 5 minutes, repeat this operation twice to remove residual ethanol, and let it air dry. Add 20 μl of DEPC water to dissolve the RNA, and use an Eppendorf nucleic acid concentration analyzer to determine the RNA concentration and purity. The RNA purity requires an OD260 / 230nm value greater than 1.80. Then, quantitative PCR was performed to detect the mRNA levels of von Willebrand factor (vWF), intercellular adhesion molecule-1 (ICAM1), thrombomodulin (THBD), and tissue factor pathway inhibitor (TFPI) using an ABI real-time quantitative PCR instrument. The quantitative PCR primers are as follows:

[0046] THBD: reserve-primer (5'-AGCACTTGTGTTGTCTGGTGGT-3') and forward-primer (5'-TGTGCACACAGAGATAGCATGAA-3') (PCR amplification conditions: denaturation 95℃ 20s; annealing 55℃ 20s; extension 72℃ 20s; 40 cycles);

[0047] vWF: reserve-primer (5'-AGCCTTTGAAACTGAAGCAT-3') and forward-primer (5'-GCCCTGGTTGCCATTGTAATTC-3') (PCR amplification conditions: denaturation 95℃ 20s; annealing 56℃ 20s; extension 72℃ 20s; 40 cycles);

[0048] ICAM1: reserve-primer (5'-ATGCCCAGACATCTGTGTCC-3') and forward-primer (5'-GGGGTCTCTATGCCCAACAA-3') (PCR amplification conditions: denaturation 95℃ 20s; annealing 55℃ 20s; extension 72℃ 20s; 40 cycles);

[0049] TFPI: reserve-primer (5'-CAAGAATGTCTGAGGGCATGTAAA-3') and forward-primer (5'-CTGCTTCTTTTTTTTTTTTTTGGTTT-3') (PCR amplification conditions: denaturation 95℃ 20s; annealing 54℃ 20s; extension 72℃ 20s; 40 cycles);

[0050] GAPDH: reserve-primer (5'-GGAGCGAGATCCCTCCAAAAT-3' and forward-primer (5'-GGCTGTTGTCATACTTCTCATGG-3) (PCR amplification conditions: denaturation 95℃ 20s; annealing 57℃ 20s; extension 72℃ 20s; 40 cycles).

[0051] See test results Figure 1 Panthioethylamine can reverse the IL-1β-induced procoagulant phenotype. IL-1β increases the expression of the procoagulant molecule vWF mRNA in vascular endothelial cells ( Figure 1 (A) and simultaneously reduced the expression of anticoagulant molecules THBD and TFPI. Figure 1 (B and C in the text). Panthiophene treatment significantly reversed these IL-1β-induced changes and downregulated vWF expression ( Figure 1 A in the middle), while raising THBD and TFPI ( Figure 1 (B and C in the text).

[0052] Next, the phosphorylation levels of NF-κB p65 (Ser536) and IkB were detected by Western blotting. The degree of phosphorylation was determined by comparing the gray values ​​of phosphorylated protein kinases with the gray values ​​of their respective total protein kinases; values ​​are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical significance. The specific steps are as follows:

[0053] To elucidate the molecular mechanism by which panthioethylamine promotes the anticoagulant phenotype of vascular endothelial cells, its effect on the NF-κB pathway was assessed. EA.Hy926 cells were pretreated with 2 × 10⁶ cells of panthioethylamine. 5Cells were seeded in 6-well plates at a concentration of 100 μM / ml. When confluence reached 80%-90%, the cells were pretreated with 100 μM tebuconazole for 12 hours (with an equal volume of 0.01 M PBS at pH 7.2-7.4 as a control), followed by IL-1β treatment (50 ng / ml) for 30 minutes. After treatment, the cells were washed once with PBS, and 0.2 ml of RIPA lysis buffer was added to each well for lysis. Lysis was performed on ice for 30 minutes at 16000 rpm × 4℃ × 15 min. The supernatant was collected, and BCA protein was quantified. The supernatant protein was denatured at 95℃ for 5 minutes with 5× loading buffer at a volume ratio, aliquoted, and stored at -20℃. Based on the BCA quantification results, samples were loaded, and SDS-PAGE gels were prepared according to the protein molecular weight. The gels were then separated by constant voltage (80-120V) gel electrophoresis and electrophoresis at a constant current of 300mA for 70-120 minutes to transfer the gels to PVDF membranes (Millipore, USA). 5% (w / w) skim milk powder was blocked in TBST (pH 7.4, 10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20) at room temperature with shaking for 1 hour. Then, 5% (w / w) of primary antibodies prepared with BSA-TBST were added: rabbit anti-NF-κB (i.e., NF-κB, Yuantai Bio, catalog number P20661), rabbit anti-phospho-NF-κB (i.e., p-NF-κB, Yuantai Bio, catalog number P20282), rabbit anti-phospho-IκB (i.e., p-IκB, MCE, catalog number HY-P80466), rabbit anti-IκB (i.e., IκB, Huayuan Bio, catalog number HY-P80466), or rabbit anti-β-actin (i.e., β-actin, Abmart, catalog number P30002S). The mixture was incubated overnight at 4°C. Rinse three times with TBST for 10 minutes each time, add horseradish peroxidase-labeled secondary antibody: goat anti-rabbit-HRP (abcam, catalog number ab6721), incubate at room temperature for 60 minutes, then add ECL (Pierce) chemiluminescent substrate for color development. Analyze using a chemiluminescence imaging analyzer; compare the gray values ​​of phosphorylated protein kinases with the gray values ​​of their respective total protein kinases to determine the degree of phosphorylation (Image J software).

[0054] Figure 2 In the figure, A represents the phosphorylation levels of NF-κB and IκB detected by Western blotting. The results showed that treatment of endothelial cells with panthioethylamine significantly inhibited the phosphorylation of NF-κB and IκB. Figure 2Figures B and C in the graphs are statistical graphs comparing the gray values ​​of phosphorylated protein kinases with the gray values ​​of their respective total protein kinases, obtained using ImageJ software, to determine the phosphorylation levels of NF-κB and IκB. The results show that panthioethylamine can significantly inhibit the phosphorylation of the NF-κB complex and its upstream regulator, IκB. Values ​​are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical significance.

[0055] Then, the ROS levels of EA.Hy926 cells pretreated with ethylparaben and exposed to IL-1β (final concentration 50 ng / ml) were detected using the DCFH-DA reactive oxygen species (ROS) fluorescent probe (Solepro, catalog number D6470-25mg). Prior to treatment, EA.Hy926 cells were injected at a concentration of 1×10⁻⁶ mg / ml. 5 Cells were seeded in 12-well plates at a concentration of 1 / ml. When confluence reached 60%-70%, the cells were pretreated with 100 μM thioethylamine for 12 hours (with an equal volume of 0.01M PBS at pH 7.2-7.4 as a control), followed by treatment with 50 ng / ml IL-1β for 30 minutes. After treatment, the cells were washed once with PBS, and a DCFH-DA reactive oxygen species (ROS) fluorescent probe (5 μmol / L) was added to each well. The cells were incubated at 37°C for 30 minutes, then washed three times with serum-free culture medium to thoroughly remove any probes that had not entered the cells. The cells were observed and photographed using a fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The scale bar for representative ROS staining images of EA.hy926 cells is 60 µm. Values ​​are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical significance.

[0056] See results Figure 3 , Figure 3 Figure A shows a representative DCFH-DA probe fluorescence image taken by fluorescence microscopy of EA.hy926 cells pretreated with panthioethylamine and with added IL-1β. The scale bar is 60 µm. The results show that the fluorescence of EA.hy926 cells in the panthioethylamine treatment group is weaker. Figure 3 B in the figure uses a fluorescence spectrophotometer to quantitatively detect the fluorescence intensity of cells in different treatment groups, showing that the fluorescence of EA.hy926 cells in the panthioethylamine treatment group is significantly reduced; Figure 3In the figure, C represents the mean DCF fluorescence intensity of cells in different treatment groups, i.e., the ROS level of the cells, detected by flow cytometry. The results showed that panthioethylamine significantly reduced the ROS level of EA.hy926 cells. Values ​​are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistical significance.

[0057] The results of this embodiment indicate that panthioethylamine exerts its anticoagulant effect by inhibiting ROS production and the NF-κB pathway, thereby promoting the synthesis of THBD and TFPI, while inhibiting the production of vWF in vascular endothelial cells.

[0058] Example 2: Endothelial cells pretreated with panthioethylamine showed significantly reduced platelet adhesion, activation, and aggregation.

[0059] To investigate the effects of pan-thioethylamine treatment on platelet function, this invention co-cultured platelets with pan-thioethylamine-pretreated human umbilical vein hybrid cells EA.hy926, and then evaluated platelet adhesion, activation, and aggregation. Specifically, in this section, EA.Hy926 cells were pretreated with pan-thioethylamine at a concentration of 1×10⁻⁶ cells. 5 The cells were seeded in 12-well plates at a concentration of / ml. When the confluence was 60%-70%, the cells were pretreated with 100 μM panthioethylamine for 12 hours (with an equal volume of physiological saline as a control). The treated cells were then co-incubated with platelets at a volume ratio of 1:5 for 1 hour, and then adherent and non-adherent platelets were collected.

[0060] First, the platelet adhesion of different treatment groups was studied. The specific steps were as follows: Adhering and non-adhering platelets collected in the panthioethylamine group and the control group were washed with PBS (centrifuged at 500g for 10 minutes at room temperature) and collected. 200μL of washed platelets (2x10⁻¹) were taken from each group. 7 The platelet-coated fibrinogen (MCE, catalog number HY-NP166, final concentration 20 mg / μL) was gently added to the slide. The plate was statically incubated at 37°C for 20, 40, and 60 minutes, respectively. After incubation, it was fixed with 4% PFA at room temperature for 15 minutes, washed twice with PBS, treated with 0.1% Triton X-100 for 5 minutes, and washed twice with PBS. The platelet-coated fibrinogen was then incubated with phalloidin (Solepro, catalog number CA1610) in the dark for 1 hour, washed twice with PBS, the PBS was discarded, and the slide was mounted. Finally, representative images of platelets adhering to fibrinogen and labeled with phalloidin were obtained using confocal microscopy. Platelet diffusion area (μm) was measured. 2 Data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01.

[0061] See results Figure 4 Platelets co-incubated with endothelial cells pretreated with panthioethylamine showed significantly reduced adhesion to fibrinogen at different time points.

[0062] Next, the platelet-rich plasma (platelet concentration of 5 × 10⁻⁶) was treated with different concentrations of the platelet activator arachidonic acid (final concentrations of 0, 200 μg / ml and 500 μg / ml) to detect platelet-rich plasma (platelet concentration of 5 × 10⁻⁶). 12 Platelet activation was assessed by evaluating platelet P-selectin expression after plasma collection ( / ml). The specific steps were as follows: Adhering and non-adhering platelets from the panthioethylamine group and the control group collected in the above steps were washed with PBS (centrifuged at 500g for 10 minutes at room temperature) and collected. 200μL of washed platelets (concentration of 5×10⁻⁶) were taken from each group. 12 The platelets were treated with different concentrations of arachidonic acid (final concentrations of 0, 200 μg / ml and 500 μg / ml) for 10 minutes each. Then, 2 μL each of flow cytometry antibodies were added: PerCP / Cyanine 5.5-tagged anti-CD41 (BioLegend, catalog number 303720) as a platelet surface marker and PE-conjugated anti-CD62P (BioLegend, catalog number 304906) as a platelet activation marker. The plates were incubated in the dark for 30 minutes. Finally, 300 μL of 4% paraformaldehyde fixative was added to each flow cytometry tube, mixed, and then analyzed by flow cytometry. The platelet activation status of different groups and different treatments was evaluated by statistically analyzing the proportion of CD41-positive and CD62P-positive activated platelets.

[0063] See results Figure 5 , Figure 5 In the graph, A represents a scatter plot of flow cytometry results, showing the proportion of P-selectin-positive platelets determined by flow cytometry. Figure 5 In the figure, B represents the platelet activation of each group of platelets after stimulation with different concentrations of arachidonic acid and co-incubation with endothelial cells pretreated with panthioethylamine, which was significantly lower than that of the control group.

[0064] Next, platelet aggregation was measured using a transilluminance aggregation assay. The specific steps were as follows: Adhering and non-adhering platelets from the panthioethylamine group and the control group collected in the above steps were washed with PBS (centrifuged at 500g for 10 minutes at room temperature) and collected. 200 μL of each washed platelet (5 × 10⁻⁶) was then taken. 12Platelets were pretreated with arachidonic acid (final concentration 500 μg / ml) for 10 minutes. The pretreated platelets were then added to a test cup equipped with a stir bar and incubated at 37°C for 3 minutes. The maximum platelet aggregation rate was measured using a platelet aggregometer. The control group received the same volume of physiological saline. Data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01.

[0065] See results Figure 6 When platelet-activating stimulant arachidonic acid was used for treatment, the platelet aggregation rate of endothelial cells pretreated with panthioethylamine was significantly lower than that of the control group.

[0066] Example 3: Proteomics analysis of mouse serum from different treatment groups

[0067] In this experiment, male C57BL / 6J mice, weighing 18–22 g and aged 2–3 months, were purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015). After one week of acclimatization, the mice were randomly divided into a control group and a panthioethylamine group (750 mg / kg / day via tail vein injection for 28 consecutive days). The control group mice were injected with the same volume of physiological saline daily. After 28 days, serum samples from both groups were collected for proteomics analysis. Proteins with a fold change (FC) ≥2 and a P-value <0.05 were selected for further analysis. Heatmaps of the most significantly upregulated and downregulated proteins in the plasma of mice treated with panthioethylamine were plotted.

[0068] See results Figure 7 Mass spectrometry analysis revealed that, compared to the saline control group, the panthioethylamine group showed significantly decreased expression of proteins such as vWF and ICAM1, and significantly increased expression of TFPI. Based on previous studies, vWF, ICAM1, TFPI, and THBD are primarily derived from endothelial cells and play a crucial role in coagulation regulation. These results suggest that panthioethylamine may mediate its antithrombotic effect by inhibiting the production of vWF and ICAM1 in endothelial cells and upregulating TFPI expression.

[0069] Example 4: In an inferior vena cava thrombosis model, panthioethylamine inhibited platelet activation and aggregation.

[0070] In this experiment, male C57BL / 6J mice, weighing 18–22 g and aged 2–3 months, were purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015). After one week of acclimatization, the mice were randomly divided into a control group and a panthioethylamine group (750 mg / kg / day via tail vein injection for 28 consecutive days). The control group mice were injected with the same volume of physiological saline daily. After 28 days, sodium citrate anticoagulated blood was collected from both groups of mice, and platelet-rich plasma (concentration 5 × 10⁻⁶) was collected by centrifugation at 200 g / min for 5 minutes. 12 Each flow cytometry tube contained 200 μL of platelet-rich plasma ( / ml). The plasma was then treated with different concentrations of arachidonic acid (final concentrations of 0, 200 μg / ml, and 500 μg / ml) for 10 minutes. Simultaneously, 2 μL each of flow cytometry antibodies were added: PerCP / Cyanine 5.5-tagged anti-CD41 (BioLegend, catalog number 133918), a platelet surface marker, and PE-conjugated anti-CD62P (BioLegend, catalog number 148305), a platelet activation marker, and incubated in the dark for 30 minutes. Finally, 300 μL of 4% paraformaldehyde fixative was added to each flow cytometry tube, and after mixing, the platelet activation level was assessed by flow cytometry. The proportions of CD41-positive and CD62P-positive activated platelets were statistically analyzed to evaluate the platelet activation levels in different groups and treatments. Data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01. from Figure 8 As shown in A, flow cytometry revealed a significant decrease in P-selectin expression on the platelet surface in the panthioethylamine treatment group, and animal experiments suggested that panthioethylamine treatment could significantly reduce platelet activation.

[0071] Next, platelet aggregation was assessed using optical transmission electron microscopy. The specific steps are as follows: Platelet-rich plasma (concentration 5 × 10⁻⁶) was collected as described above. 12 Take 200 μL of each platelet and pretreat them with arachidonic acid (final concentration 500 μg / ml) for 10 minutes. Add the pretreated platelets to a test cup with a stir bar and incubate at 37°C for 3 minutes. Measure the maximum platelet aggregation rate using a platelet aggregator. Data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01. Figure 8 B in the figure indicates that, when platelet aggregation was assessed using the light transmission aggregation method, the platelet aggregation rate was significantly reduced in the panthioethylamine group, suggesting that panthioethylamine can inhibit platelet aggregation.

[0072] Example 5: In an animal model of inferior vena cava thrombosis, panthioethylamine exhibited significant antithrombotic efficacy.

[0073] In this experiment, male C57BL / 6J mice, weighing 18–22 g and aged 2–3 months, were purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015). After one week of acclimatization, the mice were randomly divided into a control group and a panthioethylamine group (750 mg / kg / day via tail vein injection for 28 consecutive days), while the control group received the same volume of saline daily. The efficacy of panthioethylamine was then evaluated using an inferior vena cava ligation model. This invention utilizes an internationally recognized inferior vena cava thrombosis model characterized by blood stasis. The specific preparation process is as follows: Mice were fasted for 12 hours prior to surgery, but water intake was unrestricted. After anesthesia took effect, routine skin preparation, disinfection, and draping were performed. An abdominal incision was made in the midline of the mouse's abdomen to access the surgical field. The small intestine was retracted to the left side of the surgical field to fully expose the inferior vena cava. The retroperitoneum was opened, and the ligation point of the inferior vena cava below the left renal vein was identified. The inferior vena cava was carefully dissected, and the branches of the inferior vena cava below the left renal vein were exposed and ligated one by one with sutures down to the level of the iliac vein. After ligating the branches of the inferior vena cava, a suture was threaded through the ligation point. Another suture was taken and placed parallel to the main trunk of the inferior vena cava. After tying a square knot, the parallel sutures were removed, completing the inferior vena cava "stenosis" model. Postoperatively, the animals' activity, food intake, and water intake were observed. During the experimental period, the animals' survival was monitored, and the formation of inferior vena cava thrombosis in the model group mice was observed. Two days later, the inferior vena cava and attached thrombi were collected from each group of mice, and the thrombus length was measured. Pathological examination: Inferior vena cava tissue specimens were collected at the above time points, fixed in 4% paraformaldehyde for 24 hours, dehydrated, routinely embedded in paraffin, stained with hematoxylin and eosin, and observed for thrombus formation under different magnifications. Representative images of paraffin-embedded sections of the inferior vena cava stained with hematoxylin and eosin (HE) are provided. The ratio of thrombus area to lumen area in mice pretreated with panthioethylamine or treated with physiological saline as controls was statistically analyzed. All data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ns indicates no statistical significance.

[0074] The results are as follows Figure 9 As shown in A and B, compared with the saline-treated group, mice treated with panthiophene showed reduced thrombus burden (assessed by thrombus length) and significantly smaller cross-sectional area. The ratio of thrombus area to lumen area was significantly reduced in the panthiophene group compared with the control group. These results suggest that panthiophene treatment has a significant antithrombotic effect in a mouse model of inferior vena cava ligation.

[0075] Example 6: In a mouse model of pulmonary embolism, panthioethylamine exhibited significant antithrombotic effects.

[0076] In this experiment, male C57BL / 6J mice, weighing 18–22 g and aged 2–3 months, were purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015). After one week of acclimatization, the mice were randomly divided into a control group and a panthioethylamine group (750 mg / kg / day via tail vein injection for 28 consecutive days), while the control group received the same volume of physiological saline daily. The efficacy of panthioethylamine was then evaluated in a pulmonary embolism thrombosis model. This invention uses an internationally recognized pulmonary embolism thrombosis model. Collagen (Helena Laboratories, catalog number 5369-001080) and adrenaline (Helena Laboratories, catalog number 5369-001082) were used for detection. The specific steps are as follows: Mice were anesthetized by intraperitoneal injection of 1% pentobarbital (10 mL / kg body weight). Then, a mixture of collagen (0.3 μg / g body weight) and adrenaline (0.06 μg / g body weight) was injected into the tail vein in 100 μL of double-distilled water. Five minutes after injection, the lung was excised, fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Images of the HE-stained paraffin-embedded sections of mouse lung tissue were captured, and the proportion of the embolic area to the total lung area was calculated. The area of ​​pulmonary embolism in each field of view was calculated.

[0077] The results are as follows Figure 10 show, Figure 10 Tissue sections of A showed that, compared with mice treated with panthiophene, mice in the saline control group had larger areas of thrombus formation in the pulmonary vascular system. Figure 10 In Figure B, the pulmonary vascular thrombus area in the panthiophene group was significantly reduced compared to the control group, as statistically analyzed using ImageJ software. These results suggest that panthiophene treatment also has a significant antithrombotic effect in a mouse model of pulmonary embolism.

[0078] Example 7: Panthioethylamine treatment has good safety.

[0079] In this experiment, male C57BL / 6J mice, weighing 18–22 g and aged 2–3 months, were purchased from the Animal Medical Center of Nantong University (Animal Production License No. SCXK(Su)2024-0015). After one week of acclimatization, the mice were randomly divided into a control group and a panthioethylamine group (750 mg / kg / day via tail vein injection for 28 consecutive days). The control group mice were injected with the same volume of physiological saline daily. The body weight of the control and treatment groups was recorded every seven days, and body weight change curves for different treatment groups were plotted.

[0080] The results show that ( Figure 11 Compared with the control group, the panthiophene treatment group did not experience any adverse reactions, and their weight did not change significantly.

Claims

1. The use of panthioethylamine in the preparation of drugs for the prevention and / or treatment of thrombotic diseases or for the preparation of drugs that inhibit platelet activation or regulate vascular endothelial cell function.

2. The application according to claim 1, characterized in that, The panthioethylamine comprises a pharmaceutically acceptable salt, solvate, or prodrug.

3. The application according to claim 1, characterized in that, The drug is a single-ingredient preparation or a compound preparation.

4. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, sustained-release preparations, controlled-release preparations, oral liquids, injections, or lyophilized powder injections.

5. The application according to claim 1, characterized in that, The thrombotic diseases include deep vein thrombosis, pulmonary embolism, arterial thrombosis, cerebral thrombosis, or disseminated intravascular coagulation.

6. The application according to claim 1, characterized in that, The panthioethylamine exerts its antithrombotic effect by inhibiting the production of reactive oxygen species and the activation of the NF-κB signaling pathway.

7. The application according to claim 1, characterized in that, The panthioethylamine regulates coagulation balance by downregulating the expression of von Willebrand factor (vWF) and upregulating the expression of tissue factor pathway inhibitor TFPI and / or thrombomodulin THBD. Preferably, the panthioethylamine exerts its antithrombotic effect by inhibiting platelet adhesion, aggregation and / or activation.

8. The application according to claim 1, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers, diluents, or excipients.

9. A pharmaceutical composition, characterized in that, It contains a therapeutically effective amount of panthioethylamine and one or more pharmaceutically acceptable carriers.

10. The pharmaceutical composition of claim 9, characterized in that, The daily dose of panthionamide is 250 mg to 750 mg per kilogram of mice and 100 mg to 600 mg per kilogram of adults, and the panthionamide accounts for at least 20% of the mass concentration of the pharmaceutical composition.

Images