A method for improving the antithrombotic effect duration and safety of cationic polyphosphate inhibitors in vivo

Abstract

The present invention belongs to the field of medical technology, and in particular to a method for improving the antithrombotic action time and safety of cationic polyphosphate inhibitors in vivo. The present invention specifically chemically modifies cationic polyphosphate inhibitors with human serum protein ligands to improve their antithrombotic action time and safety in vivo, wherein the human serum protein ligand is 2-carboxyl phthalocyanine zinc, and the cationic polyphosphate inhibitor is low molecular weight polyethyleneimine or polylysine. Experimental data show that 2-carboxyl phthalocyanine zinc coupling significantly improves the half-life and anticoagulant action time of low molecular weight polyethyleneimine and polylysine in mice, prevents thrombosis without causing bleeding side effects, and effectively reduces the acute toxicity of low molecular weight polyethyleneimine to mice and zebrafish. Thus, it can be used to develop antithrombotic drugs with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for improving the anti-thrombotic effect time and safety of a cationic polyphosphate inhibitor in vivo. Background Art

[0002] Thrombi are mainly composed of fibrin and other components, including platelets, red blood cells, white blood cells and cholesterol crystals. Thrombosis is a major complication of cardiovascular disease, which is mainly divided into venous thrombosis (VT) and arterial thrombosis (AT). According to data from the World Health Organization (WHO), in 2019, stroke was the second leading cause of death in the world (11%), second only to ischemic heart disease (16%). Specifically, thrombosis is a common underlying mechanism for acute and major diseases such as myocardial infarction, ischemic stroke and venous thromboembolism. A quarter of the world's deaths are related to thrombosis, and the top three causes of death from cardiovascular and cerebrovascular diseases are all related to thrombosis. Therefore, thrombotic diseases have become the leading cause of death among the global population.

[0003] Thrombotic diseases result from an imbalance in the procoagulant, anticoagulant, and fibrinolytic processes. Currently, clinical medications used to prevent and treat thrombotic diseases include anticoagulants, antiplatelet drugs, and thrombolytics, with anticoagulants comprising the majority. Current clinically used antithrombotic drugs inhibit thrombosis and are associated with common adverse reactions such as bleeding. Therefore, there is an urgent need to develop novel antithrombotic drugs with a lower bleeding risk. In recent years, a variety of molecular targets for antithrombotic effects have been discovered. Among these antithrombotic targets, polyphosphate (polyP) is unique because it is an inorganic polymer, not a biomolecule. PolyP does not directly participate in the coagulation system and is not an essential component of the final common pathway of the coagulation cascade. Instead, it promotes coagulation by accelerating certain coagulation processes. Therefore, targeting PolyP is highly likely to avoid the bleeding events associated with traditional antithrombotic drugs.

[0004] PolyP has a high tendency to promote vascular coagulation. It is not only a highly effective activator of coagulation factor XII (FXII), but also accelerates the activation of coagulation factor V, counteracting the effects of anticoagulant drugs. In addition, PolyP can be incorporated into fibrin clots to change the fibrin network, stabilize the blood clot and resist thrombolysis. There are also reports that PolyP can inhibit the fibrinolytic system by accelerating thrombin-mediated activation of thrombin-activated fibrinolysis inhibitor (TAFI).

[0005] The most commonly used method for inhibiting PolyP is to neutralize the negative charge of PolyP using polycationic compounds, polymers, and proteins. According to literature reports, low-molecular-weight polyethylenimine (LMW-PEI) and poly-L-lysine (PLL) bind most strongly to PolyP on a mass and molar basis, respectively. These inhibitors effectively prolong thrombosis in a FeCl3-induced mouse arterial thrombosis model. However, in vivo, these low-molecular-weight cationic polymers are readily cleared by the kidneys and have an extremely short in vivo retention time (half-life of approximately several minutes), severely limiting the duration of their pharmacological effects. Furthermore, PLL, a positively charged polypeptide, is easily degraded by proteases, while PEI exhibits high cytotoxicity and acute in vivo toxicity.

[0006] To improve the in vivo retention time and long-term efficacy of LMW-PEI and PLL, reduce cytotoxicity, and improve biocompatibility, the present invention chemically modifies LMW-PEI and PLL with 2-carboxyl zinc phthalocyanine (ZnPc), coupling ZnPc to the amino groups of LMW-PEI and PLL to obtain ZnPc-LMW-PEI and ZnPc-PLL. ZnPc is a high-affinity targeting molecule for the human serum protein HSA. Therefore, ZnPc-LMW-PEI and ZnPc-PLL can bind to human serum albumin (HSA) in the blood circulation, reducing renal metabolism and increasing in vivo retention time. At the same time, it reduces contact with hydrolytic enzymes and cells, reducing the risk of hydrolysis and in vivo toxicity. The present invention is the first to use ZnPc to modify the cationic inhibitor of polyphosphate and utilize the targeting effect of zinc phthalocyanine on HSA, thereby reducing contact with serum components and blood cells, solving the toxicity problems of cationic compounds and the short metabolic cycle in the body, and demonstrating excellent anti-thrombotic activity both in vivo and in vitro. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for improving the antithrombotic effect duration and safety of cationic polyphosphate inhibitors in vivo. Taking PolyP as the target and based on the advantage of serum albumin as a drug delivery carrier, the cationic inhibitors LMW-PEI and PLL of polyphosphate are modified by utilizing the targeting effect of zinc phthalocyanine derivatives on serum albumin, thereby reducing their biological toxicity, achieving a long-lasting antithrombotic effect, and obtaining a safe and long-lasting antithrombotic drug.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention relates to the use of a human serum protein ligand in the preparation of a drug for antithrombotic treatment of a subject by improving the antithrombotic effect duration and safety of a cationic polyphosphate inhibitor, wherein the human serum protein ligand is 2-carboxyphthalocyanine zinc, and the cationic polyphosphate inhibitor is a low molecular weight polyethyleneimine or polylysine.

[0010] Furthermore, the above application includes the following steps:

[0011] S1: Weigh 2-carboxyphthalocyanine zinc and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, add N,N-dimethylformamide and N,N-diisopropylethylamine, and react under magnetic stirring in a 40°C water bath in the dark for 30 minutes to obtain an activation solution;

[0012] S2: Weigh low molecular weight polyethyleneimine, add N,N-dimethylformamide thereto, and repeatedly pipette to uniformly disperse it to obtain an N,N-dimethylformamide solution containing low molecular weight polyethyleneimine;

[0013] S3: adding the activated solution obtained in step S1 dropwise to the N,N-dimethylformamide solution containing low molecular weight polyethyleneimine obtained in step S2, and reacting with magnetic stirring for 24 hours in a water bath at 40°C in the dark; after the reaction, removing the N,N-dimethylformamide by rotary evaporation under reduced pressure to obtain a solid crude product;

[0014] S4: dissolving the crude product obtained in step S3 with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a primary freeze-dried product; dissolving the primary freeze-dried product with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a secondary freeze-dried product, which is the drug ZnPc-LMW-PEI for antithrombotic treatment;

[0015] The ZnPc-LMW-PEI has improved antithrombotic effect duration and safety compared to low molecular weight polyethyleneimine;

[0016] The molar ratio of the 2-carboxyl phthalocyanine zinc to the low molecular weight polyethyleneimine is 1:1.

[0017] Furthermore, the above application includes the following steps:

[0018] S1: Weigh 2-carboxyphthalocyanine zinc and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, add N,N-dimethylformamide and N,N-diisopropylethylamine, and react under magnetic stirring in a 40°C water bath in the dark for 30 minutes to obtain an activation solution;

[0019] S2: Weigh poly-lysine, add N,N-dimethylformamide to it, and repeatedly pipette to disperse it evenly to obtain an N,N-dimethylformamide solution containing poly-lysine;

[0020] S3: adding the activated solution obtained in step S1 dropwise to the N,N-dimethylformamide solution containing polylysine obtained in step S2, and reacting under magnetic stirring in a 40°C water bath in the dark for 24 hours; after the reaction, removing the N,N-dimethylformamide by rotary evaporation under reduced pressure to obtain a solid crude product;

[0021] S4: dissolving the crude product obtained in step S3 with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a primary freeze-dried product; dissolving the primary freeze-dried product with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a secondary freeze-dried product, which is the drug ZnPc-PLL for antithrombotic treatment;

[0022] The ZnPc-PLL has improved antithrombotic effect duration and safety compared to polylysine;

[0023] The molar ratio of the 2-carboxyl phthalocyanine zinc to polylysine is 1:1.

[0024] The present invention primarily utilizes polyphosphate (PolyP) as a drug receptor and polymers (ZnPc-LMW-PEI and ZnPc-PLL) to create a pharmaceutical preparation, addressing the toxicity and short metabolic cycle of cationic compounds. In vitro biological activity experiments on the preparation revealed that ZnPc-LMW-PEI and ZnPc-PLL significantly prolonged their in vivo half-lives. In vitro binding experiments, measuring the binding of ZnPc-LMW-PEI and ZnPc-PLL to human serum albumin, revealed that the preparation exhibited an excellent in vivo active half-life. Furthermore, the modified polyphosphate inhibitor reduced the risk of in vivo toxicity and platelet aggregation induction, providing a theoretical basis for the potential clinical use of the preparation. In vitro coagulation experiments using PolyP demonstrated that the drugs, both before and after ZnPc modification, significantly inhibited the procoagulant effects of polyphosphate in vitro. Experiments in a ferric chloride-induced carotid artery embolism model in mice revealed that ZnPc-LMW-PEI and ZnPc-PLL promoted thrombolysis and significantly prolonged the time required for thrombus formation to obstruct blood flow, demonstrating their effectiveness as PolyP inhibitors. Bleeding risk assessment experiments revealed that the ZnPc-modified PolyP inhibitors exhibited no significant bleeding risk, exhibited a low bleeding risk, and were relatively safe in vivo. Therefore, ZnPc-LMW-PEI and ZnPc-PLL may be high-affinity PolyP inhibitors with long-lasting action and high biocompatibility, potentially protecting blood vessels from inflammation and thrombosis.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention is the first to start from a small molecule polyphosphate inhibitor and, by modifying it to reduce toxicity and simultaneously utilizing the combination of HSA and ligand molecules to carry out long-term transformation, it greatly expands the research scope of polyphosphate inhibitors and provides new ideas for the development of new polyphosphate inhibitors.

[0027] (2) ZnPc-LMW-PEI and ZnPc-PLL have the function of inhibiting polyphosphate targets, promoting thrombolysis, and can play a therapeutic role in thrombotic diseases.

[0028] (3) In the safety evaluation, the bleeding risk of ZnPc-LMW-PEI and ZnPc-PLL was verified, proving that while they have anti-thrombotic effects, they do not increase their bleeding risk. In addition, the drugs have a long half-life in the body and excellent biocompatibility. Therefore, it is expected that the occurrence of side effects can be reduced while reducing the dosage, providing new ideas for the development of new anti-thrombotic drugs without bleeding risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : In Example 2, by changing the concentration of HSA, the changes in the fluorescence intensity of ZnPc-LMW-PEI and ZnPc-PLL were measured.

[0030] Figure 2 : Graph showing the in vivo retention time of ZnPc-LMW-PEI and ZnPc-PLL measured by relative blood concentrations at various time points after tail vein administration in Example 3.

[0031] Figure 3 : Graph showing the effects of ZnPc-LMW-PEI and ZnPc-PLL on the clotting time of platelet-poor plasma induced by different concentrations of PolyP in Example 4.

[0032] Figure 4 : In Example 5, the inhibitory effects of ZnPc-LMW-PEI and ZnPc-PLL on FeCl3-induced carotid artery thrombosis in mice were determined, i.e., the carotid artery occlusion time of mice after FeCl3 stimulation 10 minutes after administration (A) and the carotid artery occlusion time of mice after FeCl3 stimulation 2 hours after administration (B) are compared.

[0033] Figure 5 : In Example 6, the effects of ZnPc-LMW-PEI and ZnPc-PLL on the bleeding time (A) and bleeding volume (B) of mice were evaluated using the mouse tail-chopping model.

[0034] Figure 6 : Comparison of the in vivo toxicity survival rates of various concentrations of ZnPc-LMW-PEI and ZnPc-PLL on zebrafish evaluated in Example 7.

[0035] Figure 7 : Graph showing the toxic effects of ZnPc-LMW-PEI and ZnPc-PLL on EA.hy 926 cell line (human umbilical vein cell fusion cells) in Example 8.

[0036] Figure 8 : Infrared spectra of ZnPc, ZnPc-LMW-PEI and ZnPc-PLL. ZnPc curve: 1690cm -1 (C=O stretching vibration of carboxyl group); ZnPc-LMW-PEI curve: 1614 cm -1 (Stretching vibration of amide bond C=O) 2950 cm -1 (Saturated CH stretching vibration) ZnPc-PLL curve: 1640 cm -1 (Stretching vibration of amide bond C=O) 2932 cm -1 (saturated CH stretching vibration). DETAILED DESCRIPTION

[0037] For a better understanding of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the scope of protection claimed in the present invention is not limited to the scope of the examples. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. In the following examples, the various processes and methods not described in detail are conventional methods well known in the art.

[0038] Example 1: Preparation of ZnPc-LMW-PEI and ZnPc-PLL

[0039] The preparation steps of ZnPc-LMW-PEI are as follows:

[0040] S1: Use a precision balance to weigh 19.23 mg of 2-carboxyphthalocyanine zinc (ZnPc, which was prepared according to the method published in our laboratory (Chen JC, Chen NS, Huang JF, et al., Inorg. Chem. Commun. [J], 2006, 9: 313–315) and 46.77 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) into a round-bottom reaction flask. Add 2.5 mL of N,N-dimethylformamide (DMF) and 65 μL of N,N-diisopropylethylamine (DIEA). Incubate the mixture in a dark place in a 40°C water bath with magnetic stirring for 30 min to fully activate the carboxyl groups of ZnPc, thereby obtaining an activated solution.

[0041] S2: Using a precision balance, weigh 24.61 mg of low molecular weight polyethyleneimine (LMW-PEI, purchased from Aladdin, product number P121256, CAS number 25987-06-8, with a degree of polymerization of 1.5 and an average molecular weight of 800 Da). Add 2.5 mL of DMF and pipette repeatedly to disperse the mixture evenly to obtain a DMF solution containing the LMW-PEI.

[0042] S3: The activated solution obtained in step S1 was added dropwise to the DMF solution containing LMW-PEI obtained in step S2, and the mixture was reacted with magnetic stirring for 24 hours in a dark place and a 40°C water bath. After the reaction, the DMF was removed by vacuum rotary evaporation to obtain a solid crude product.

[0043] S4: The crude product obtained in step S3 was dissolved in 5 mL of ultrapure water, centrifuged at 10,000 rpm for 1-2 times, each time for 10 minutes, the supernatant was collected and pre-frozen at -80°C for 2 hours, and then placed in a freeze dryer at -70°C for freeze-dried for 48 hours to obtain a primary freeze-dried product; to fully remove insoluble impurities, the primary freeze-dried product was dissolved in 5 mL of ultrapure water, centrifuged at 10,000 rpm for 1-2 times, each time for 10 minutes, the supernatant was collected and pre-frozen at -80°C for 2 hours, and then placed in a freeze dryer at -70°C for freeze-dried for 48 hours to obtain a secondary freeze-dried product, namely ZnPc-LMW-PEI.

[0044] The preparation steps of ZnPc-PLL are as follows:

[0045] S1: Use a precision balance to weigh 19.23 mg of 2-carboxyphthalocyanine zinc (ZnPc) and 46.77 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) into a round-bottom reaction flask. Add 2.5 mL of N,N-dimethylformamide (DMF) and 65 μL of N,N-diisopropylethylamine (DIEA). Incubate the mixture in a dark place in a 40°C water bath with magnetic stirring for 30 min to fully activate the carboxyl groups of ZnPc and obtain an activated solution.

[0046] S2: Using a precision balance, weigh 92.3 mg of poly-lysine (PLL, purchased from SIGMA, product number P0879, CAS number 25988-63-0, with a degree of polymerization of 10-30 and an average molecular weight of 1000-5000). Add 2.5 mL of DMF and pipette repeatedly to disperse the PLL to obtain a DMF solution.

[0047] S3: adding the activated solution obtained in step S1 dropwise to the DMF solution containing PLL obtained in step S2, and reacting with magnetic stirring for 24 hours in a dark place and a 40°C water bath; after the reaction, removing the DMF by vacuum rotary evaporation to obtain a solid crude product;

[0048] S4: The crude product obtained in step S3 was dissolved in 5 mL of ultrapure water, centrifuged at 10,000 rpm for 1-2 times, each time for 10 minutes, the supernatant was collected and pre-frozen at -80°C for 2 hours, and then placed in a freeze dryer for freeze-drying at -70°C for 48 hours to obtain a primary freeze-dried product; to fully remove insoluble impurities, the primary freeze-dried product was dissolved in 5 mL of ultrapure water, centrifuged at 10,000 rpm for 1-2 times, each time for 10 minutes, the supernatant was collected and pre-frozen at -80°C for 2 hours, and then placed in a freeze dryer for freeze-drying at -70°C for 48 hours to obtain a secondary freeze-dried product, namely ZnPc-PLL.

[0049] The structural formula of ZnPc-LMW-PEI is as follows:

[0050] Wherein, n represents the degree of polymerization of low molecular weight polyethyleneimine, which is 1.5.

[0051] The structural formula of ZnPc-PLL is as follows:

[0052] In the formula, n represents the degree of polymerization of polylysine, which is an integer between 7 and 30.

[0053] The infrared spectra of ZnPc, ZnPc-LMW-PEI and ZnPc-PLL are shown in Figure 8 .

[0054] Example 2: Nonlinear fitting curve of the KD value of ZnPc-LMW-PEI / ZnPc-PLL binding to HSA based on the ZnPc fluorescence signal

[0055] A 0.09 mg / mL ZnPc-LMW-PEI solution, a 0.05 mg / mL ZnPc-PLL solution, and a 4 mM HSA stock solution were prepared in PBS buffer (0.01 M, pH 7.4). The HSA stock solution was then serially diluted two-fold to 10 concentrations. A 96-well black plate was plated, and 20 μL of HSA solution of varying concentrations was added to each well. An equal volume of ZnPc-LMW-PEI solution or ZnPc-PLL solution was then added, and PBS buffer (0.01 M, pH 7.4) was added to a final volume of 100 μL per well. After mixing thoroughly, the mixture was incubated at 37°C for 10 minutes. Finally, the fluorescence intensity (ex = 610 nm, em = 680 nm) of the ZnPc-LMW-PEI or ZnPc-PLL was measured using a microplate reader, and the fluorescence emission spectrum (ex = 610 nm, em = 640-750 nm) was scanned. The obtained data were fitted with nonlinearity using GraphPad Prime 8.0 to calculate the KD value of its binding to HSA. Each test was repeated at least 3 times.

[0056] The results are as follows Figure 1 As shown in the figure, the KD values ​​of ZnPc-LMW-PEI and ZnPc-PLL for HSA are 37.88μM and 64.60μM, respectively. This indicates that although the introduction of LMW-PEI and PLL reduced the affinity of ZnPc for HSA (KD = 0.9μM), ZnPc-LMW-PEI and ZnPc-PLL were still able to bind to HSA relatively strongly. One possible reason for the weakened binding force is that the introduction of LMW-PEI and PLL increased steric hindrance. However, from the perspective of drug delivery, the relative reduction in the dissociation constant of drug binding to HSA can enable greater drug release at the target site.

[0057] Example 3: Determination of the In Vivo Retention Time of ZnPc-LMW-PEI or ZnPc-PLL To investigate whether ZnPc-coupled compounds could successfully prolong the in vivo retention time of LMW-PEI and PLL, we injected a certain concentration of the coupled compound into the tail vein of mice and used the blood circulation to study the in vivo retention time of the coupled compound. ZnPc itself has a good fluorescence signal, which facilitates the monitoring of ZnPc concentrations in plasma at different time points. To address the issue that LMW-PEI and PLL do not inherently have fluorescent properties, we introduced FITC to modify them and used them as a control group with the ZnPc-coupled group. Twenty healthy male ICR mice aged 6 to 8 weeks were randomly divided into four groups of five mice each. Four groups of mice were injected via the tail vein with FITC-LMW-PEI (200 μM), FITC-PLL (200 μM), ZnPc-LMW-PEI (50 μM), and ZnPc-PLL (50 μM), respectively. Each mouse received an injection volume of 160 μL. At 2, 5, 10, 20, 30, 1, 2, 4, 8, 12, and 24 hours after injection, 40 μL of blood was collected by tail puncture (EP tubes were pre-moistened with sodium citrate solution, and 5 μL of sodium citrate anticoagulant was added). 10 μL of supernatant plasma was added to 90 μL of buffer (the crosslinking buffer for FITC-LMW-PEI and FITC-PLL is formulated as follows: 7.56 g NaHCO₃, 1.06 g Na₂CO₃, 7.36 g NaCl dissolved in 800 mL of deionized water, adjusted to pH 9.0, and then diluted to 1 L with deionized water; the buffer for ZnPc-LMW-PEI and ZnPc-PLL is a 10% SDS solution). Mix thoroughly, and measure the fluorescence intensity of FITC (ex = 490 nm, em = 525 nm) or ZnPc (ex = 610 nm, em = 680 nm) using a microplate reader. Data were processed using Graphpad Prism 8.0 software.

[0058] The results are as follows Figure 2 As shown in Figure 3 . Calculations show that the in vivo retention times of ZnPc-LMW-PEI and ZnPc-PLL are 2 and 35 times longer than those of LMW-PEI and PLL, respectively. These results indirectly demonstrate the feasibility of utilizing ZnPc to bind to endogenous serum albumin to increase the in vivo retention time of PolyP inhibitors.

[0059] Example 4: In vitro antithrombotic activity of ZnPc-LMW-PEI and ZnPc-PLL

[0060] Venous blood was drawn from healthy volunteers and centrifuged at 3000 rpm for 15 min at room temperature. The upper plasma was collected to obtain platelet-poor plasma (PPP). Inorganic polyphosphate (polyP) was extracted with reference [Pokhrel A, Lingo JC, Wolschendorf F, Gray MJ. Assaying for Inorganic Polyphosphate in Bacteria. J VisExp. 2019 Jan 21; (143): 10.3791 / 58818.], and the extracted PolyP powder was prepared into a 1 mg / mL stock solution with buffer (pH 7.4 25 mM Tris-HCl, 150 mM NaCl), and then diluted two-fold to 10 concentrations; at the same time, different concentrations of PolyP inhibitor solutions were prepared with buffer: LMW-PEI solution 0.5 mg / mL, PLL solution 0.5 mg / mL, ZnPc-LMW-PEI solution 0.9 mg / mL, and ZnPc-PLL solution 0.6 mg / mL. To each well of a 96-well transparent plate, add 10 μL of PolyP solution of varying concentrations, 10 μL PolyP inhibitor solution, 40 μL buffer, and 30 μL PPP. Finally, add 10 μL of 150 mM CaCl₂ solution. Immediately place the plate in a microplate reader and monitor the coagulation curve of each well at 405 nm. The monitoring temperature is 37°C, the monitoring time is 40 minutes, and the monitoring interval is 30 seconds. Three replicates are performed for each PolyP concentration. A control group without PolyP inhibitor is set up, and a buffer group is set up as a blank control group.

[0061] The results are as follows Figure 3As shown. When the concentration of Escherichia coli-derived PolyP is ≥0.0078 mg / mL, it can significantly shorten the coagulation time and show a significant procoagulant effect. When a certain concentration of PolyP inhibitor is present, the procoagulant effect of PolyP is eliminated within a certain concentration range, and we found that ZnPc-LMW-PEI and ZnPc-PLL obtained after ZnPc modification still maintain good PolyP inhibition properties. In addition, 0.05 mg / mL LMW-PEI and 0.09 mg / mL ZnPc-LMW-PEI have a stronger inhibitory effect on the procoagulant effect of PolyP than 0.05 mg / mL PLL and 0.06 mg / mL ZnPc-PLL.

[0062] Example 5: Thrombolytic Effects of ZnPc-LMW-PEI and ZnPc-PLL in FeCl3-Induced Mouse Carotid Artery Embolism Model

[0063] Healthy male ICR mice were randomly divided into five groups of eight: saline, LMW-PEI, PLL, ZnPc-LMW-PEI, and ZnPc-PLL. After tail vein injection of the drug, the mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate (5 μL / g). After disinfection, the left common carotid artery was surgically dissected and exposed. The artery was separated from other tissues using filter paper. Blood flow in the common carotid artery was monitored using a laser speckle blood flow imager. Under a microscope, a small amount of 12% FeCl₃ solution was dripped onto the common carotid artery, and the duration of occlusion of the common carotid artery was recorded. The drug dosage for each group was as follows: 5 mg / kg body weight (BW) for the LMW-PEI and PLL groups, 9 mg / kg BW for the ZnPc-LMW-PEI group, and 6 mg / kg BW for the ZnPc-PLL group. In addition, to investigate whether the coupling of ZnPc can achieve the long-term antithrombotic effect of PolyP inhibitors, 2 hours after tail vein administration, the left common carotid artery of mice was stimulated with 12% FeCl3 solution, and the above steps were repeated to monitor the blood flow in the common carotid artery of mice and record the vascular occlusion time.

[0064] The results are as follows Figure 4As shown. Except for the PLL group, the LMW-PEI, ZnPc-LMW-PEI, and ZnPc-PLL groups were able to significantly prolong the carotid artery occlusion time in mice 10 minutes after administration. Compared with the saline group, the carotid artery occlusion time in the LMW-PEI, ZnPc-LMW-PEI, and ZnPc-PLL groups was prolonged by 3.3 times, 2.6 times, and 2.8 times, respectively, demonstrating a good antithrombotic effect. The PLL group did not show a good antithrombotic effect 10 minutes after administration, which may be due to its short in vivo residence time. The ZnPc-PLL group was still able to significantly prolong the carotid artery occlusion time in mice 2 hours after administration. Compared with the saline group, the carotid artery occlusion time in mice was prolonged by 1.8 times, demonstrating a good antithrombotic effect, indicating that the coupling of ZnPc achieves a long-term effect of PLL's antithrombotic effect.

[0065] Example 6: Evaluation of bleeding risk by tail bleeding experiment

[0066] Using a mouse tail-clip bleeding model, we analyzed the bleeding time and volume at the initial hemostasis stage to assess whether hemostasis is impaired and whether bleeding risk exists after tail vein injection of a PolyP inhibitor. Male ICR mice, 6 to 8 weeks of age and in good condition, were randomly divided into five groups of six. Each group received tail vein injections of saline, LMW-PEI (5 mg / kg body weight), PLL (5 mg / kg body weight), ZnPc-LMW-PEI (9 mg / kg body weight), or ZnPc-PLL (6 mg / kg body weight). Ten minutes after tail vein administration, mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate (5 μL / g). The tails were then clipped 10 mm from the tip of the tail. Immediately after tail clipping, the tails were immersed in preheated (37°C) isotonic saline. Blood was collected, and the bleeding time was recorded until the first hemorrhage stopped. After the bleeding is complete, blood cells are collected by centrifugation (3000 rpm for 10 min), the supernatant is removed, and 2 mL of red blood cell lysis buffer (8.3 g / L NH4Cl, 1.0 g / L KHCO3, 0.037 g / L EDTA) is added to the pellet. Lyse the cells overnight at 4°C in the dark. After thorough mixing, the lysed solution is centrifuged at 10,000 rpm for 5 min. 100 μL of the supernatant is transferred to a 96-well transparent plate and its absorbance at 575 nm is measured. Each blood sample is repeated in triplicate.

[0067] The results are as follows Figure 5As shown. Tail vein injection of LMW-PEI significantly increased the bleeding time and amount of bleeding in mice, indicating a significant bleeding risk. However, ZnPc-modified LMW-PEI significantly shortened the bleeding time and amount of bleeding caused by LMW-PEI in mice, reducing the bleeding risk of LMW-PEI. Although ZnPc-LMW-PEI increased the bleeding time of the mouse tail compared to the control group, the amount of bleeding did not increase significantly, indicating overall hemostatic safety. In addition, neither PLL nor ZnPc-PLL showed significant bleeding side effects.

[0068] Example 7: In Vivo Toxicity of ZnPc-LMW-PEI and ZnPc-PLL in Zebrafish Fry. Drug stock solutions of varying concentrations were prepared in E3 medium: LMW-PEI 1.5 mg / mL, PLL 6 mg / mL, ZnPc-LMW-PEI 2.7 mg / mL, ZnPc-PLL 7.2 mg / mL, aspirin (ASN) 300 μM, and adrenalin hydrochloride (AH) 180 μM in 0.03% DMSO. Zebrafish larvae, normally developed to day 3, were plated in 12-well plates, with 18 per well. For the LMW-PEI group, the LMW-PEI stock solution was serially diluted two-fold with E3 medium to 12 concentrations. To each well, 1 mL of LMW-PEI solution of varying concentrations, 1 mL of E3 medium containing 0.03% DMSO, and 1 mL of AH solution were added. Other drugs were administered as in the LMW-PEI group, except that LMW-PEI was replaced with the corresponding drug. The model group received 1 mL of E3 medium, 1 mL of AH solution, and 1 mL of E3 medium containing 0.03% DMSO, respectively. The blank control group received only E3 medium and 0.01% DMSO. Following drug administration, zebrafish larvae were kept in the dark for 16 hours. The number of larval deaths after 16 hours was counted, and survival curve analysis was performed.

[0069] The results are as follows Figure 6 The LD values ​​of LMW-PEI, ZnPc-LMW-PEI, PLL, and ZnPc-PLL were calculated. 50 According to LD 50 It can be seen that the toxicity of the four PolyP inhibitors to zebrafish is LMW-PEI>ZnPc-LMW-PEI>ZnPc-PLL>PLL from high to low.

[0070] Example 8: CCK-8 Detection of Cytotoxicity of ZnPc-LMW-PEI and ZnPc-PLL The cell suspension was diluted with DMEM medium and EA.hy 926 cells were seeded in a 96-well cell culture plate at 6000 cells per well in a volume of 100 μL per well. The cells were then cultured in a cell culture incubator (37°C, 5% CO2) for 24 h to allow the cells to adhere. Sterile water and sterile tubes were used to prepare the stock solutions of each sample. The stock solutions of LMW-PEI, PLL, ZnPc-LMW-PEI, and ZnPc-PLL were 7.2 mg / mL, 6 mg / mL, 10.1 mg / mL, and 6.8 mg / mL, respectively. The stock solutions were diluted 3-fold with fresh medium to a total of 10 concentrations. Remove the 96-well plate and discard the old culture medium. Add 100 μL of diluted PolyP inhibitor at different concentrations to each well. Repeat for four replicates for each concentration. Set up a blank control group and continue incubating the cells in the incubator for 24 hours. After 24 hours, aspirate the old culture medium and add 100 μL of PBS buffer to each well to wash away the inhibitor and suspended cells. Next, add 100 μL of fresh culture medium containing 10% CCK-8 reagent to each well and incubate in the incubator for 1-2 hours until color development is complete. After color development, measure the absorbance of each well at 450 nm on a microplate reader. Then, calculate cell viability.

[0071] The results are as follows Figure 7 As shown in the figure, the cytotoxicity of LMW-PEI and ZnPc-LMW-PEI on EA.hy 926 cells was concentration-dependent. When the concentration of ZnPc-LMW-PEI was 1.12 mg / mL, the cell viability was 81.6%, while the cell viability was only 54.8% when 0.8 mg / mL of LMW-PEII was added. Similarly, the cell viability (89.3%) after adding 0.37 mg / mL of ZnPc-LMW-PEI was higher than the cell viability (57.9%) after adding 0.27 mg / mL of LMW-PEI. PLL and ZnPc-PLL showed no cytotoxicity, and the cell viability remained around 80% at the highest concentration.

[0072] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Use of ZnPc-LMW-PEI in the preparation of a drug for antithrombotic treatment of a subject by increasing the duration and safety of the antithrombotic effect of a cationic polyphosphate inhibitor, characterized in that: The cationic polyphosphate inhibitor is a low molecular weight polyethyleneimine; the structural formula of the ZnPc-LMW-PEI is: , n is 1.

5.

2. The use according to claim 1, characterized in that: The preparation method of the ZnPc-LMW-PEI comprises the following steps: S1: Weigh 2-carboxyphthalocyanine zinc and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, add N,N-dimethylformamide and N,N-diisopropylethylamine, and react under magnetic stirring in a 40°C water bath in the dark for 30 minutes to obtain an activation solution; S2: Weigh low molecular weight polyethyleneimine, add N,N-dimethylformamide thereto, and repeatedly pipette to uniformly disperse it to obtain an N,N-dimethylformamide solution containing low molecular weight polyethyleneimine; S3: adding the activated solution obtained in step S1 dropwise to the N,N-dimethylformamide solution containing low molecular weight polyethyleneimine obtained in step S2, and reacting with magnetic stirring for 24 hours in a water bath at 40°C in the dark; after the reaction, removing the N,N-dimethylformamide by rotary evaporation under reduced pressure to obtain a solid crude product; S4: dissolving the crude product obtained in step S3 with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a primary freeze-dried product; dissolving the primary freeze-dried product with ultrapure water, centrifuging, collecting the supernatant, and placing it in a freeze dryer for freeze drying to obtain a secondary freeze-dried product, which is the drug ZnPc-LMW-PEI for antithrombotic treatment; The ZnPc-LMW-PEI has improved antithrombotic effect duration and safety compared to low molecular weight polyethyleneimine.

3. The use according to claim 2, characterized in that: The molar ratio of the 2-carboxyl phthalocyanine zinc to the low molecular weight polyethyleneimine is 1:1.

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