Antagonistic activity of two types of water-soluble porous organic polymers towards heparin-like anticoagulants

Abstract

The present invention belongs to the field of biomedicine and heparin anticoagulant drug antagonism, and specifically relates to two types of water-soluble cationic porous organic polymers (SOFs and POPs) with low toxicity that can effectively antagonize unfractionated heparin and low molecular weight heparin.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and heparin anticoagulant drug antagonism, and specifically relates to the application of two types of water-soluble positive ion porous organic polymers in the antagonism of unfractionated heparin and low molecular weight heparin. Background Art

[0002] Heparin is a linear sulfated glycosaminoglycan composed of repeating units of α-1,4-linked uronic acid and D-glucosamine, and has the highest negative charge density among biological macromolecules.

[0003]

[0004] Heparin binds strongly to antithrombin and significantly enhances its ability to inhibit the activity of serine protease coagulation factors (primarily factor Xa and thrombin) in the coagulation cascade. Heparin has been widely used clinically as an anticoagulant since 1935. Its primary clinical applications include the treatment of thromboembolism; prophylactic treatment of patients at high risk of embolism; postoperative prophylaxis of thromboembolism; and prevention of coagulation and thrombosis due to circulatory system interventions (such as cardiovascular diagnostic procedures, catheterization, and cardiac and vascular surgery), as well as numerous other procedures (such as extracorporeal circulation, the use of artificial organs, and organ transplantation). Clinically, the anticoagulant effect of heparin must be neutralized or reversed after these procedures to prevent bleeding problems caused by the continued anticoagulant effect of residual heparin.

[0005] Heparin drugs used in clinical practice include unfractionated heparin (UFH, extracted from porcine intestinal mucosa or bovine lung, with an average molecular weight of approximately 15kDa), low molecular weight heparins (LMWHs, small molecule fragments with an average molecular weight of approximately 3.6–6.5kDa obtained by depolymerization of unfractionated heparin), and fondaparinux (molecular weight 1.7kDa), which is a modified synthesis of the natural pentose structure contained in both UFH and LMWHs. However, in clinical practice, overdose is often caused by the difficulty in controlling heparin dosage and individual differences among patients, resulting in side effects such as bleeding, hyperkalemia, systemic allergic reactions (including angioedema), osteoporosis, and thrombocytopenia.

[0006]

[0007] To date, protamine (average molecular weight approximately 3–4.5 kDa) is the only FDA-approved heparin antagonist. It is a protein rich in arginine groups extracted from mature sperm of fish (such as salmon and herring). The common sequence is 32 amino acids, and positively charged arginine accounts for 67% of the total sequence.

[0008] Currently, protamine cannot be produced through chemical synthesis, so its production is limited by fish supplies and poses a risk of contamination. Furthermore, protamine can cause serious adverse reactions, including anaphylactic shock, systemic hypotension, severe pulmonary vasoconstriction, and idiopathic fatal cardiac arrest. Serious complications occur in 2.6% of cardiac surgeries treated with protamine, and up to 10% of patients undergoing this treatment experience some form of problem. Furthermore, protamine cannot distinguish between active and inactive heparin sequences, neutralizing only approximately 60% of various low-molecular-weight heparins. In some cases, this antagonism may fail, leading to further anticoagulation issues. However, no clinically approved protamine alternatives have yet been developed.

[0009] Therefore, there is an urgent need in the art to develop antagonists of heparin-type anticoagulant drugs that are simple, easy to obtain, highly effective, rapid, and less toxic, for use as a substitute for protamine. Summary of the Invention

[0010] The purpose of the present invention is to provide a use of cationic porous organic polymer SOFs and POPs for antagonizing heparin anticoagulant drugs.

[0011] The first aspect of the present invention provides the use of cationic porous organic polymers SOFs and POPs for preparing an antagonist pharmaceutical composition for heparin anticoagulants;

[0012] The porous organic polymer SOFs are formed by self-assembly of tetrahedral molecules and cucurbit[8]uril (CB[8]) as shown in the following formula I:

[0013]

[0014] The porous organic polymer POPs is formed by cross-linking tetrahedral molecules through intermolecular [2+2] olefin dimerization cycloaddition reaction as shown in the following formula II:

[0015]

[0016] Wherein, R is a substituted or unsubstituted C1-C4 alkyl group, or a substituted or unsubstituted C1-C4 alkoxy group; wherein the substituent is selected from the following group: OH, NH2.

[0017] In another preferred embodiment, in formula I, R is selected from the following group: OCH2CH2NH2, OCH3;

[0018] In another preferred embodiment, in formula II, R is selected from the following group: CH3, CH2CH2OH, CH2CH2NH2, CH2CH(OH)CH2OH.

[0019] In another preferred embodiment, the cucurbit[8]uril (CB[8]) has a structure as shown below:

[0020]

[0021] In another preferred embodiment, the porous organic polymer SOFs and POPs are three-dimensional structures in a diamond configuration constructed based on tetrahedral molecules.

[0022] In another preferred embodiment, the porous organic polymer SOFs are formed by self-assembly of tetrahedral molecules containing aryl pyridinium salt groups and cucurbit[8]uril (CB[8]) main molecules in an aqueous phase.

[0023] In another preferred embodiment, the porous organic polymer SOFs are formed by self-assembly of tetrahedral molecules A1 or A2 and cucurbit[8]uril (CB[8]) main molecules in aqueous phase:

[0024]

[0025] In another preferred embodiment, the porous organic polymer POPs is formed by cross-linking tetrahedral molecules containing styrene groups through intermolecular [2+2] olefin dimerization cycloaddition reaction.

[0026] In another preferred embodiment, the porous organic polymer POPs is formed by crosslinking tetrahedral molecules selected from the group consisting of B1, B2, B3, and B4 through intermolecular [2+2] olefin dimerization cycloaddition reaction.

[0027]

[0028] In another preferred embodiment, the molecular weight of the porous organic polymer SOFs is 230-269 kD.

[0029] In another preferred embodiment, the molecular weight of the porous organic polymer POPs is 64-74 kD.

[0030] In another preferred embodiment, the surfaces and internal cavities of the porous organic polymer SOFs and POPs are positively charged and uniformly dispersed.

[0031] In another preferred embodiment, the heparin anticoagulant drug is selected from the following group: unfractionated heparin, low molecular weight heparin, or a combination thereof.

[0032] In another preferred embodiment, the average molecular weight of the unfractionated heparin is about 15 kDa.

[0033] In another preferred embodiment, the average molecular weight of the low molecular weight heparin is about 3.6-6.5 kDa.

[0034] In another preferred embodiment, the low molecular weight heparin is selected from the group consisting of dalteparin sodium, enoxaparin sodium, nadroparin calcium, or a combination thereof.

[0035] In another preferred embodiment, the heparin anticoagulant drug is selected from the following group: unfractionated heparin, dalteparin sodium, enoxaparin sodium, nadroparin calcium, or a combination thereof.

[0036] In another preferred embodiment, the antagonistic ratios of SOFs and POPs to heparin are 0.835 mg / 100 IU and 1.05 mg / 100 IU, respectively.

[0037] In a second aspect of the present invention, a pharmaceutical composition or preparation for antagonizing heparin anticoagulants is provided, the pharmaceutical composition or preparation comprising:

[0038] (a) cationic porous organic polymer SOFs and / or POPs as active ingredients; and

[0039] (b) a pharmaceutically acceptable carrier;

[0040] The pharmaceutical composition or preparation is used to antagonize heparin anticoagulant drugs.

[0041] In another preferred embodiment, component (a) accounts for 0.001-99.99 wt% of the total weight of the preparation; preferably 0.01-99.9 wt%; more preferably 0.05-90 wt%.

[0042] In another preferred embodiment, the pharmaceutically acceptable carrier is a physiological saline solution.

[0043] In another preferred embodiment, the pharmaceutical composition or preparation is in the form of an injection, tablet, capsule, pill, suspension or emulsion.

[0044] In another preferred embodiment, a method for antagonizing heparin-type anticoagulant drugs is provided, characterized in that the method comprises: administering a therapeutically effective amount of a water-soluble cationic porous organic polymer, or a pharmaceutical composition or preparation thereof, to a subject in need.

[0045] In another preferred embodiment, a method for in vitro non-therapeutic antagonism of heparin-type anticoagulants is provided, characterized in that the method comprises the steps of: contacting a water-soluble cationic porous organic polymer with a subject in need in vitro, thereby achieving in vitro antagonism of heparin-type anticoagulants.

[0046] In a third aspect of the present invention, there is provided a use of a composition of a cationic porous organic polymer and protamine for preparing an antagonist of heparin anticoagulants.

[0047] In a fourth aspect of the present invention, a kit is provided, comprising:

[0048] (1) a first container, and a first pharmaceutical composition in the first container, wherein the first pharmaceutical composition comprises a first compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and / or

[0049] (2) a second container, and a heparin-based anticoagulant drug located in the second container, wherein the heparin-based anticoagulant drug is selected from unfractionated heparin, low molecular weight heparin, or a combination thereof; and / or

[0050] (3) an nth container, and an nth pharmaceutical composition located in the nth container, wherein the nth pharmaceutical composition comprises an nth compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; wherein n is any positive integer from 3 to 8;

[0051] Wherein, the first compound and the nth compound are both polymers composed of tetrahedral molecules selected from the following group:

[0052]

[0053] and / or (4) optional instructions for use.

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The isothermal calorimetric titration spectra of SOF1 (0.05 mM, saline solution) with heparin molecules (0.4 mM, saline solution) such as a) unfractionated heparin, b) dalteparin sodium, c) enoxaparin sodium, and d) nadroparin calcium are shown.

[0056] Figure 2 The isothermal calorimetric titration spectra of POP1 (0.05 mM, saline solution) with heparin molecules (0.4 mM, saline solution) such as a) unfractionated heparin, b) dalteparin sodium, c) enoxaparin sodium, and d) nadroparin calcium are shown.

[0057] Figure 3 The isothermal calorimetric titration spectra of protamine (0.05 mM, saline solution) with heparin molecules (0.4 mM, saline solution) such as a) unfractionated heparin, b) dalteparin sodium, c) enoxaparin sodium, and d) nadroparin calcium are shown.

[0058] Figure 4The fluorescence titration spectra of heparin molecules (1.0 mM, PBS solution) such as unfractionated heparin (a), dalteparin sodium (b), enoxaparin sodium (c), and nadroparin calcium (d) added dropwise to SOF1 solution (0.005 mM, PBS solution) are shown.

[0059] Figure 5 The fluorescence titration spectra of heparin molecules (1.0 mM, PBS solution) such as unfractionated heparin (a), dalteparin sodium (b), enoxaparin sodium (c), and nadroparin calcium (d) added dropwise to POP1 solution (0.005 mM, PBS solution) are shown.

[0060] Figure 6 The results of activated partial thromboplastin time (aPTT) experiments in which SOF1, SOF2, protamine, etc. antagonize (a) unfractionated heparin (2.48 IU / mL), low molecular weight heparin (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL) in human plasma are shown.

[0061] Figure 7 The results of the activated partial thromboplastin time (aPTT) experiment of POP1, POP2, POP3, POP4, protamine, etc. antagonizing (a) unfractionated heparin (2.48 IU / mL), low molecular weight heparin (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), (d) nadroparin calcium (1.76 IU / mL) in human plasma are shown.

[0062] Figure 8 The results of activated partial thromboplastin time (aPTT) experiments in bovine plasma show that SOF1, SOF2, protamine, etc. antagonize (a) unfractionated heparin (2.48 IU / mL), low molecular weight heparin (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL).

[0063] Figure 9 The results of the activated partial thromboplastin time (aPTT) experiment of POP1, POP2, POP3, POP4, protamine, etc. in bovine plasma antagonizing (a) unfractionated heparin (2.48 IU / mL), low molecular weight heparin (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL) are shown.

[0064] Figure 10The results of experiments showing SOF1 and POP1 antagonizing the anti-factor Xa activity of (a) unfractionated heparin (2.48 IU / mL) and (b) low molecular weight heparin (using dalteparin sodium as an example, 2.52 IU / mL) in human plasma are shown.

[0065] Figure 11 The thromboelastography results of SOF1 (0.25 mg / mL, 0.50 mg / mL) and (a) unfractionated heparin (2.48 IU / mL), (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL) in a human whole blood environment are shown.

[0066] Figure 12 The thromboelastography test results of SOF2 (0.45 mg / mL, 0.75 mg / mL) and (a) unfractionated heparin (2.48 IU / mL), (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL) in a human whole blood environment are shown.

[0067] Figure 13 The thromboelastography test results of POP1 (0.13 mg / mL, 0.26 mg / mL) and (a) unfractionated heparin (2.48 IU / mL), (b) dalteparin sodium (2.52 IU / mL), (c) enoxaparin sodium (2.56 IU / mL), and (d) nadroparin calcium (1.76 IU / mL) in a human whole blood environment are shown.

[0068] Figure 14 Shown are the results of an in vivo mouse tail transection experiment demonstrating SOF1 and POP1 antagonism to heparin. (a) Cartoon illustration of the mouse tail transection experiment; (b) Bleeding time and (c) bleeding volume corresponding to the unfractionated heparin (248 IU / kg) antagonism experiment; (d) Bleeding time and (e) bleeding volume corresponding to the dalteparin (252 IU / kg) antagonism experiment. All models were administered twice intravenously, with SOF1 and POP1 doses of 3.34 mg / kg and 2.6 mg / kg, respectively. The saline group served as the blank control, the unfractionated heparin or dalteparin groups served as the negative control, and the unfractionated heparin-protamine group served as the positive control. Data are mean ± standard error (n = 10). *p < 0.05, ***p < 0.01, ****p < 0.005; ns indicates "no significant difference" between experimental and control groups. SEM: standard error of the mean.

[0069] Figure 15Results of in vivo heparin antagonism experiments in rats are shown. (a) Cartoon model of the in vivo aPTT experiment in rats; (b) Changes in aPTT clotting time as a function of in vivo metabolic time for the blank control group (normal saline) and the negative control group (unfractionated heparin or dalteparin sodium); (c) Changes in aPTT clotting time as a function of in vivo metabolic time for SOF1 (3.3 mg / kg), POP1 (2.6 mg / kg), and protamine against unfractionated heparin (248 IU / kg); (d) Changes in aPTT clotting time as a function of in vivo metabolic time for SOF1, POP1, and protamine against dalteparin sodium (248 IU / kg). Blood was collected at 0, 5, 7.5, 10, 20, 35, and 60 minutes, respectively. All models were administered intravenously twice, and data are mean ± standard error (n = 10).

[0070] Figure 16 Shown are the results of hemolysis experiments of SOFs and POPs on (a) human red blood cells and (b) rat red blood cells.

[0071] Figure 17 Shown are the results of CCK-8 cytotoxicity assays of SOF1 on (a) H9C2 cells and (b) MCF-7 cells.

[0072] Figure 18 The results of CCK-8 cytotoxicity experiments of SOF2 on (a) H9C2 cells and (b) MCF-7 cells are shown.

[0073] Figure 19 Shown are the results of CCK-8 cytotoxicity experiments of POP1 on (a) H9C2 cells and (b) MCF-7 cells.

[0074] Figure 20 Shown are the results of CCK-8 cytotoxicity assays of POP2 on (a) H9C2 cells and (b) MCF-7 cells.

[0075] Figure 21 Shown are the results of CCK-8 cytotoxicity assays of POP3 on (a) H9C2 cells and (b) MCF-7 cells.

[0076] Figure 22 Shown are the results of CCK-8 cytotoxicity experiments of POP4 on (a) H9C2 cells and (b) MCF-7 cells.

[0077] Figure 23The results of acute toxicity tests in mice are shown. Following injection of different doses of SOF1 (3.3, 15.7, 31.3, and 62.6 mg / kg), (a) changes in mouse body weight over a 15-day observation period, (b) organ indices, and (c) serum urea nitrogen and alanine aminotransferase levels over a 15-day observation period. Following injection of different doses of POP1 (2.6, 5.2, and 10.4 mg / kg), (d) changes in mouse body weight over a 15-day observation period, (e) organ indices, and (f) serum urea nitrogen and alanine aminotransferase levels over a 15-day observation period were shown. Each group consisted of 10 mice, half male and half female. A blank control group received saline.

[0078] Figure 24 The results of pathological sections of major organs are shown 15 days after injection of different doses of SOF1 (3.3, 15.7, 31.3, 62.6 mg / kg) and a saline control group.

[0079] Figure 25 The results of pathological sections of major organs 15 days after injection of different doses of POP1 (2.6, 5.2, 10.4 mg / kg) and a saline control group are shown. DETAILED DESCRIPTION

[0080] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that two types of water-soluble, cationic porous organic polymers, SOFs and POPs, have antagonistic effects on heparin-based anticoagulants. Further experiments demonstrated that these two types of water-soluble, cationic porous organic polymers, SOFs and POPs, can antagonize unfractionated heparin and low-molecular-weight heparin, while also exhibiting high biosafety. This work led to the completion of the present invention.

[0081] The in vitro activity experiments in this invention were conducted using poor platelet plasma (PPP) and whole blood. Animal plasma, such as bovine or ovine plasma, can also be used for this assay. Human PPP and whole blood were obtained from blood samples approved for scientific research by the Shanghai Blood Center.

[0082] the term

[0083] Heparin-type anticoagulants

[0084] Heparin is a linear sulfated glycosaminoglycan composed of repeating units of α-1,4-linked uronic acid and D-glucosamine, and has the highest negative charge density among biological macromolecules.

[0085] Heparin can highly bind to antithrombin and significantly enhance the ability of antithrombin to inhibit the activity of serine protease coagulation factors (mainly coagulation factor Xa and thrombin) during the blood coagulation cascade.

[0086] Heparin drugs used clinically include unfractionated heparin (UFH, extracted from porcine intestinal mucosa or bovine lungs, with an average molecular weight of approximately 15kDa), low molecular weight heparins (LMWHs, small molecule fragments with an average molecular weight of approximately 3.6–6.5kDa obtained by depolymerization of unfractionated heparin), and fondaparinux (molecular weight 1.7kDa), which is a modified synthesis of the natural pentose structure contained in both UFH and LMWHs.

[0087] Active ingredient

[0088] As used herein, the terms "compounds of the present invention" and "active ingredients of the present invention" are used interchangeably to refer to two types of water-soluble cationic porous organic polymers, SOFs and POPs. It should be understood that the term also includes mixtures of the above components.

[0089] The present invention also includes pharmaceutically acceptable salts of cationic porous organic polymer SOFs and POPs. The term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salt formed by the polymer of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0090] The cationic porous organic polymer SOFs and POPs of the present invention can be prepared by methods well known to those skilled in the art, with no particular limitation on the reaction parameters of each step. Furthermore, the typical compounds of the present invention can also be obtained commercially.

[0091] As used herein, in cationic porous organic polymer SOFs and POPs, if a chiral carbon atom exists, the chiral carbon atom may be in R configuration, S configuration, or a mixture of the two.

[0092] Pharmaceutical compositions and methods of administration

[0093] The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients or carriers. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-100 mg per dose. Preferably, "one dose" is one capsule or tablet.

[0094] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0095] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration, or subcutaneous administration).

[0096] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0097] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0098] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally used in the art, water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures thereof.

[0099] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0100] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0101] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0102] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0103] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 5 to 100 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0104] The main advantages of the present invention are:

[0105] (1) SOFs and POPs of the present invention are water-soluble nanomaterials with advantages such as positive charge (adjustable charge density), porosity (adjustable pore size), high water solubility, high stability, and low toxicity;

[0106] (2) SOFs and POPs can be used to bind to guest molecules in a multivalent manner and absorb negative ionic heparin anticoagulants through synergistic electrostatic interactions, thereby achieving efficient antagonism or neutralization of heparin anticoagulants by SOFs and POPs.

[0107] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0108] Example 1: Preparation of water-soluble cationic organic polymer SOFs and POPs

[0109] Preparation of SOFs

[0110] The water-soluble cationic organic polymer SOF1-2 of the present invention is prepared according to the document Nat. Commun. 2014, 5: 5574. The preparation process is as follows:

[0111]

[0112] SOF1 is a three-dimensional extended stereo network structure with good solubility in aqueous solution (based on the concentration of monomers, [A n ]=30mM, [CB[8]]=15mM), it can be regarded as a nanoparticle with an approximately spherical shape, and its hydrated particle diameter is 67.3nm. According to Materials Studio software, the crystal package ( α=β=γ=90), it can be deduced that its number of repeating units is n=63.4, its molecular weight is about 264.9kD, and its charge number is 253.6. The aqueous solution of SOF1 has a maximum UV absorption at 458nm.

[0113] SOF2 is a three-dimensional extended stereo network structure with good solubility in aqueous solution (based on the concentration of monomers, [A n ]=10mM,[CB[8]]=5mM), it can be regarded as a nanoparticle with an approximately spherical shape and a hydrated particle diameter of 65.0nm. α=β=γ=90°), it can be deduced that its number of repeating units is n=61.1, its molecular weight is about 230.5kD, and its charge number is 244.4. The aqueous solution of SOF2 has a maximum UV absorption at 458nm.

[0114] 1.2. Preparation of POPs

[0115] The water-soluble cationic porous organic polymer POP1-4 of the present invention is prepared according to the document ACS Appl. Mater. Interfaces 2020, 12, 1404-1411. The preparation process is as follows:

[0116]

[0117] POP1-4 is a three-dimensional extended stereo network structure with good solubility in aqueous solution (based on the concentration of monomers, [B n ]=50mM), it can be regarded as a nanoparticle with a nearly spherical shape, and its hydrated particle diameter is 37.2nm. α=β=γ=90°), it can be deduced that the number of repeating units is 36.5, the molecular weight is about 63.8-72.6kD, and the charge number is 291.7. The aqueous solution of POP1-4 has a maximum UV absorption at 425nm.

[0118] Example 2: Binding of SOFs and POPs to different heparin molecules in solution

[0119] The concentration of SOFs is based on a tetrahedral monomer molecule binding to two cucurbit[8]urils, the concentration of POPs is based on a tetrahedral monomer molecule, and the concentration of heparin is based on a repeating unit consisting of α-1,4 linked uronic acid and D-glucosamine. This experiment takes the binding of SOF1 and POP1 to heparin as an example, using physiological saline as the medium, preparing SOF1 and POP1 (50μM), and heparin molecular solution (0.4mM), and conducting isothermal calorimetric titration experiments and fluorescence titration experiments. Figure 1-3 As shown in Table 1, we found that the apparent binding constant between heparin and SOF1 or POP1 was 10 6 M -1level, and has a strong binding effect on heparin molecules. Figure 4 As shown in the fluorescence titration experiment, with the addition of heparin molecules, the fluorescence intensity of SOF1 gradually decreased until it no longer changed, and the fluorescence quenching could be as high as 69%. The fluorescence intensity of POP1 gradually increased until it no longer changed, and the fluorescence enhancement could be as high as 843% ( Figure 5 These results indicate that SOF1 or POP1 of the present invention has a strong binding effect on heparin molecules and stable absorption.

[0120] Table 1. Thermodynamic parameters of SOF-C2N and POP1 binding to heparin in saline medium (25°C)

[0121]

[0122] superscript 1 、 2 、 3 Respectively represent the binding effects of SOF1, POP1 and protamine with different heparin molecules.

[0123] Example 3: In vitro antagonistic activity study of SOFs and POPs

[0124] The in vitro activity tests of the present invention include: activated partial thromboplastin time (aPTT) test ( Figures 6 to 9 ), Anti-Xa factor test ( Figure 10 ), thromboelastography (TEG) test ( Figures 11 to 13 The heparin excess model used in the "PPP or whole blood-heparin-antagonist system" of the present invention is as follows: the final concentration of unfractionated heparin is 2.48 IU / mL, the final concentration of dalteparin sodium is 2.52 IU / mL, the final concentration of enoxaparin sodium is 2.56 IU / mL, and the final concentration of nadroparin calcium is 1.76 IU / mL. Among them, "PPP or whole blood-normal saline" is a blank control, "PPP or whole blood-heparin-normal saline" is a negative control, and "PPP or whole blood-heparin-protamine" is a positive control.

[0125] (1) aPTT experiment

[0126] The present invention uses a fully automated coagulation analyzer (UP3000, Shanghai Sun Biotechnology) to perform the aPTT test. The "PPP-heparin-antagonist system" is automatically tested according to the manufacturer's instructions, using low-platelet plasma as the experimental medium. First, heparin or saline is mixed with low-platelet plasma in a ratio of 1:99. The resulting heparinized plasma is incubated in a 37°C water bath for 5 minutes. Then, 17 μL of SOFs, POPs, or protamine solution (dissolved in 0.9% saline) is added to 153 μL of heparinized plasma (10%, v / v) and incubated for 5 minutes before testing.

[0127] like Figure 6-9 The aPTT results shown in the figure show that the SOFs and POPs of the present invention can not only achieve complete antagonism against unfractionated heparin, but also have a wider therapeutic window; they also show better antagonism (neutralization) efficiency and a wider and more stable therapeutic window for low molecular weight heparin than protamine. For unfractionated heparin, the SOFs and POPs of the present invention can achieve complete antagonism, with a maximum antagonism rate of 91-100%. In human plasma, the neutralization efficiency of SOF1 and SOF2 against unfractionated heparin exceeds 90% in the concentration range of 0.0167-0.096 mg / mL and 0.0415-0.0943 mg / mL, respectively. Figure 6 a); The neutralization efficiency of POP1-POP4 for unfractionated heparin exceeded 90% in the following concentration ranges: 0.0210-0.07 mg / mL, 0.0150-0.0748 mg / mL, 0.0141-0.0704 mg / mL, and 0.0239-0.0796 mg / mL ( Figure 7 a). In bovine plasma, SOF1 and SOF2 also showed over 90% antagonism against unfractionated heparin in the concentration ranges of 0.0167-0.0925 mg / mL and 0.0754-0.1320 mg / mL ( Figure 8 a); The concentration ranges of POP1-POP4 that also had an antagonistic effect of more than 90% on unfractionated heparin were: 0.0210-0.0612 mg / mL, 0.0224-0.0561 mg / mL, 0.0161-0.0503 mg / mL, and 0.0318-0.0597 mg / mL ( Figure 9 a). For low molecular weight heparin, in human plasma, the antagonistic rate of SOFs and POPs of the present invention to dalteparin sodium is 90-93% ( Figure 6 b, 7b), the antagonism rate to enoxaparin sodium was 76-87% ( Figure 6 c, 7c), the antagonism rate to nadroparin calcium was 81-100% ( Figure 6 d, 7d); in bovine plasma, SOFs and POPs antagonized dalteparin sodium by 91-99% ( Figure 8 b, 9b), the antagonism rate to enoxaparin sodium was 87-100% ( Figure 8 c, 9c), the antagonism rate to nadroparin calcium is 85-100% ( Figure 8 d, 9d). Taking dalteparin sodium as an example, the SOFs and POPs of the present invention also have a more stable and wider therapeutic window for low molecular weight heparin than protamine. In human plasma, the neutralization efficiency of SOF1 and SOF2 for dalteparin sodium exceeds 85% in the following concentration ranges: 0.0167-0.0835 mg / mL and 0.0453-0.132 mg / mL, respectively. Figure 6 b); The concentration ranges of POP1-POP4 for the neutralization efficiency of dalteparin sodium exceeding 85% were: 0.0210-0.0612 mg / mL, 0.0150-0.0561 mg / mL, 0.0161-0.0402 mg / mL and 0.0239-0.0497 mg / mL ( Figure 7 b) In bovine plasma, SOF1 and SOF2 also showed over 90% antagonistic efficacy against dalteparin sodium at concentrations ranging from 0.0334-0.0668 mg / mL and 0.0754-0.0943 mg / mL, respectively. Figure 8 b); The concentration ranges of POP1-POP4 with more than 90% antagonistic efficacy against dalteparin sodium were: 0.0210-0.07 mg / mL, 0.0299-0.0654 mg / mL, 0.0161-0.0805 mg / mL and 0.0398-0.0696 mg / mL ( Figure 9 b). In contrast, although protamine can also achieve complete antagonism against unfractionated heparin, the concentration window for over 90% antagonism is much narrower, at only 0.0140-0.0210 mg / mL and 0.0122-0.0306 mg / mL in human plasma and bovine plasma, respectively. Protamine has only partial antagonistic activity against low molecular weight heparins, with the highest antagonism rates against dalteparin sodium, enoxaparin sodium, and nadroparin calcium being 78%, 57%, and 69% in human plasma, respectively. Figure 6 ); in bovine plasma 81%, 61% and 69% ( Figure 8 These results indicate that protamine has a significantly lower antagonistic effect on low molecular weight heparin than SOFs or POPs. Decreasing or increasing the protamine dosage resulted in a dramatic decrease in the antagonistic effect.

[0128] The above aPTT experimental results show that the antagonism rate of SOFs and POPs of the present invention against low molecular weight heparin is significantly better than that of protamine, and they have a wider therapeutic window for both unfractionated heparin and low molecular weight heparin.

[0129] (2) Anti-Xa factor experiment

[0130] The present invention uses a commercially available anti-factor Xa kit (Biophen Heparin Anti-Xa (two-step method), Hyphen Biomed, France) to conduct anti-factor Xa experiments. Using SOF1 and POP1 as experimental models for antagonism against unfractionated heparin and dalteparin sodium, respectively, the "PPP-heparin-antagonist system" was diluted 10-fold and tested in low-platelet plasma according to the manufacturer's instructions.

[0131] like Figure 10 As shown, the experimental results show that SOF1 and POP1 of the present invention can achieve complete antagonism against unfractionated heparin, with an antagonism rate of 100%, which is similar to the antagonism rate of protamine (92%) ( Figure 10 a). SOF1 and POP1 of the present invention can also achieve high antagonism to dalteparin sodium, up to 92% and 94% respectively ( Figure 10 b). In contrast, protamine can only partially antagonize dalteparin sodium, with an antagonism rate of only 60% ( Figure 10 b) The antagonism rate calculated by the anti-Xa factor test in the present invention is consistent with the aPTT test results, confirming each other, further demonstrating that SOFs and POPs drugs have excellent antagonism against unfractionated heparin and low molecular weight heparin, and have a long therapeutic window.

[0132] (3) Thromboelastography test

[0133] The present invention used SOF1, SOF2, and POP1 as experimental models to conduct thromboelastography experiments on unfractionated heparin, dalteparin sodium, enoxaparin sodium, and nadroparin calcium. The relevant experiments were conducted in whole blood using a thromboelastograph (Haema T4 thromboelastograph).

[0134] The thromboelastography test method for neutralizing heparin with an antagonist in the present invention is as follows: 1) First, whole blood (1 mL) is mixed with 10.1 μL of heparin (248 IU / mL unfractionated heparin; 252 IU / mL dalteparin sodium; 256 IU / mL enoxaparin sodium; 176 IU / mL nadroparin calcium) or normal saline, and incubated at 37°C for 5 minutes. 2) Then, 112.2 μL of antagonist solution or normal saline is added and mixed, and the incubation is continued for 5 minutes. Referring to the amount of antagonist used to achieve the best antagonistic efficiency in the partial thromboplastin test, such as Figure 11 、 12In the elastography tests shown in Figures 1 and 2, two antagonist concentrations of SOF1, SOF2, POP1, and protamine were used. The final antagonist concentrations of SOF1 were 0.025 mg / mL and 0.050 mg / mL, the final antagonist concentrations of SOF2 were 0.045 mg / mL and 0.075 mg / mL, the final antagonist concentrations of POP1 were 0.013 mg / mL and 0.26 mg / mL, and the final antagonist concentration of protamine was 0.026 mg / mL. 3) Pipette 1 mL of the mixed whole blood into a plastic tube containing kaolin activator, invert the tube five times to mix, and then transfer 340 μL of the mixture into a cuvette preheated to 37°C (20 μL of 0.2 mol / L calcium chloride solution must have been added to the cuvette in advance). 4) Immediately begin the thromboelastography measurement for at least 60 minutes.

[0135] like Figure 11 、 12 As shown, thromboelastometry results demonstrate that SOF1 and SOF2 of the present invention, at both concentrations, can restore coagulation function to normal levels in whole blood overdosed with unfractionated heparin and low molecular weight heparins (dalteparin sodium, enoxaparin sodium, and nadroparin calcium), with parameters such as clotting time and clot strength all within normal ranges. Thromboelastography results also demonstrate that POP1 of the present invention significantly restores coagulation function with unfractionated heparin and also significantly restores coagulation function with low molecular weight heparin.

[0136] Example 4: In vivo activity studies of SOFs and POPs

[0137] The present invention takes the antagonism of SOF1 and POP1 to unfractionated heparin and dalteparin sodium as an example, studies the animal experiment of antagonist reversal of heparin activity, adopts mouse tail cross-section experiment and rat body aPTT experimental model, observes respectively the apparent bleeding situation (bleeding time, bleeding volume) caused by antagonist reversal heparin excessive and the influence (aPTT time) on intrinsic coagulation pathway.Use tail vein 2 injection administration mode, inject heparin and antagonist successively at intervals of 5 minutes, and then collect blood.The experiment is with injection "normal saline + normal saline" as blank control, "heparin + normal saline" as negative control, and "heparin + protamine" as positive control.The mouse injection drug volume is 0.1-0.2mL, and the rat injection drug volume is 0.5-1.0mL.During the experiment, anesthesia and anesthesia maintenance are carried out using isoflurane.

[0138] (1) Mouse tail transect experiment

[0139] The present invention uses ICR mice (18-22 g) as an experimental model, with 5 male and 5 female mice. At 0 minutes, heparin or saline was injected; at 5 minutes, an antagonist or saline was injected; at 8 minutes, isoflurane anesthesia was initiated; at 10 minutes, the tail of the mouse was transected 2.5 mm from the end, and blood was collected from the tail using a 15 x 15 mm filter paper. The bleeding end point of the tail blood collection was the disappearance of blood spots on the filter paper with the naked eye, and the total time did not exceed 120 minutes ( Figure 14 a) Collection intervals were set at 2 minutes per session (10-40 minutes), 5 minutes per session (41-60 minutes), 10 minutes per session (61-90 minutes), and 20 minutes per session (91-130 minutes). The blood-containing filter paper was dissolved in 2 mL of 10% NaOH solution until the filter paper was completely lysed. The supernatant was collected by centrifugation and transferred to a 96-well plate. The absorbance at 450 nm was measured using a microplate reader. The total amount of bleeding for each mouse was calculated by comparing the results to a standard curve.

[0140] like Figure 14 As shown in b, the average bleeding time and bleeding volume of the saline blank control group were 21.40±3.02 minutes and 24.22±7.45μL. The average bleeding time and bleeding volume of the unfractionated heparin negative control group were 60.80±9.56 minutes and 243.21±58.77μL, and the average bleeding time and bleeding volume of the dalteparin sodium negative control group were 64.20±9.46 minutes and 206.27±48.95μL, which were significantly different from the blank control (P<0.005). In addition, the dispersion of the negative control group was large (bleeding time was between 22-120 minutes, and total bleeding volume was between 10.36-565.82μL), indicating that the individual differences in the amount of heparin used in mice were large. Figure 14 In groups b and 14c of "unfractionated heparin_protamine", when protamine was used for antagonism, the unfractionated heparin was completely antagonized, and the average bleeding time and total bleeding volume returned to normal values ​​(29.33±6.42 minutes, 44.87±422.00 μL), which were not significantly different from those in the normal saline blank group (P=0.1753 and P=0.2065). However, the dispersion of the antagonism rate was large (bleeding time ranged from 10-80 minutes, and total bleeding volume ranged from 4.69-440.37 μL). At the same time, protamine has partial antagonistic activity against dalteparin sodium (45±5.42 minutes, 101.23±36.75 μL), which is significantly different from the normal saline blank group (P=0.1753 and P=0.2065), and also significantly different from the dalteparin sodium negative control group (P=0.25 and P=0.11), with a large dispersion (bleeding time ranged from 12-63 minutes, and total bleeding volume ranged from 6.91-373.60 μL).

[0141] In contrast, SOF1 and POP1 can achieve complete antagonism against unfractionated heparin and dalteparin sodium, and their antagonistic activities are far superior to those of protamine, with smaller dispersion ( Figure 14 b, 14c and 14d, 14e). Specifically, when SOF1 and POP1 antagonized unfractionated heparin, the average bleeding time and total bleeding volume returned to normal levels (27.60±4.50 minutes, 42.47±9.10 μL and 20.10±3.94 minutes, 31.59±13.13 μL), with no significant difference compared with the normal saline group (P=0.2919, P=0.1585 and P=0.8066, P=0.6492), but with significant difference compared with the heparin negative control group (P=0.0002, P=0.0068 and P=0.0000481, P=0.0051), and the values ​​of bleeding time and bleeding volume were more convergent ( Figure 14 b, 14c). When SOF1 and POP1 antagonized dalteparin sodium, the average bleeding time and total bleeding volume returned to normal levels (21.30±2.92 minutes, (18.93±6.75) μL and 18.20±2.46 minutes, 21.07±8.27 μL), with no significant difference compared with the normal saline group (P=0.9822, P=0.6236 and P=0.4457, P=0.7909), but with significant difference compared with the heparin negative control group (P=0.0017, P=0.0030 and P=0.0005, P=0.0033). Similarly, the values ​​of bleeding time and total bleeding volume were more convergent ( Figure 14 d, 14e).

[0142] The results of the mouse tail cross-section experiment of the present invention show that SOFs and POPs can completely antagonize unfractionated heparin and low molecular weight heparin (taking dalteparin sodium as an example) in mice, and the individual differences in the antagonistic effect are small, which has obvious advantages over protamine.

[0143] (2) aPTT experiment in rats

[0144] The present invention uses SD rats (180-220 g) as the experimental model, with 3 male and 3 female rats. The aPTT experiment in rats is performed by collecting blood samples from the jugular vein. Blood samples (total volume 0.4 mL, containing 0.05 mL 3.8% sodium citrate) are collected at 0 minutes, 5 minutes, (7.5 minutes), 10 minutes, 20 minutes, 35 minutes, and 60 minutes. During the blood collection process, the rat is placed in a supine position with the head of the rat at an angle of approximately 45 degrees to the body. The syringe needle is inserted into the jugular vein parallel to the triangular point between the shoulder and the neck of the rat to collect blood ( Figure 15a). The collected rat anticoagulated blood was centrifuged (3000 rpm, 4°C, 15 minutes) to collect the upper low-platelet plasma, and then the aPTT time was measured using an automatic coagulometer. The aPTT time remained almost unchanged within 0 minutes, with an average value of 30.76±1.30s ( Figure 15 b). After heparin injection, the aPTT time is significantly prolonged, and the anticoagulant effect within 5 minutes exceeds the instrument detection limit (maximum detection time 180s). As the metabolic time increases, the heparin content gradually decreases, and the dispersion of the residual amount of heparin in the blood is very large ( Figure 15 b). After adding antagonists SOF1 or POP1, the residual amounts of unfractionated heparin and dalteparin sodium in the blood decreased significantly within 2.5 minutes and completely returned to normal levels ( Figure 15 c and 15d). Protamine's antagonism of unfractionated heparin also restored normal levels within 2.5 minutes ( Figure 15 c). However, for dalteparin sodium, a certain amount of heparin remained within 2.5 minutes (aPTT time was 47.1s), and it returned to normal levels within 5 minutes ( Figure 15 d) This result shows that in rats, the SOFs and POPs of the present invention can achieve complete antagonism against both unfractionated heparin and low molecular weight heparin (using dalteparin sodium as an example) within 2.5 minutes, and the antagonistic activity against dalteparin sodium is higher than that of protamine.

[0145] Example 5: Biosafety Study of SOFs and POPs

[0146] The present invention studied the biosafety of SOFs and POPs through in vitro red blood cell hemolysis experiments, cytotoxicity experiments, and in vivo acute toxicity experiments. Based on the aPTT experiment, the usable concentration ranges (in human plasma) are as follows: [SOF1] = 16.7-85.3 μg / mL, [SOF2] = 41.5-132.0 μg / mL; [POP1] = 21.0-70.0 μg / mL, [POP2] = 15.0-74.8 μg / mL, [POP3] = 14.1-70.4 μg / mL, [POP4] = 23.9-79.6 μg / mL. The concentrations with the highest antagonistic rates were: [SOF1] = 16.7 μg / mL, [SOF2] = 60.3 μg / mL; [POP1] = 21.0 μg / mL, [POP2] = 15.0 μg / mL, [POP3] = 21.0 μg / mL, [POP4] = 16.1 μg / mL)

[0147] (1) Red blood cell hemolysis test

[0148] The red blood cell hemolysis experiment of the present invention was conducted on human red blood cells and rat red blood cells. Different concentration gradients of SOFs (0.69-751.58 μg / mL) and POPs (0.77-452.53 μg / mL) in physiological saline were added to a 2% red blood cell solution, followed by incubation at 37°C for 1 hour. After centrifugation (3000 rpm, 4°C, 15 minutes), the supernatant was collected and the absorbance value was measured. Distilled water was used to completely lyse the red blood cells as a positive control (100% hemolysis), and red blood cells in pure physiological saline were used as a negative control (0% hemolysis). Figure 16 As shown, the results show that the SOFs and POPs of the present invention have no hemolytic toxicity to human and rat red blood cells at high concentrations and have high safety at the concentrations used.

[0149] (2) Cytotoxicity assay

[0150] like Figure 17-22 The results showed that the half-inhibitory concentrations of SOF1 and SOF2 on the cell viability of H9C2 and MCF-7 cells were 386.8 μg / mL and 262.4 μg / mL, respectively. Figure 17 ), >339.4μg / mL and >339.4μg / mL( Figure 18 ); The half-inhibitory concentrations of POP1, POP2, POP3, and POP4 on H9C2 and MCF-7 cell viability were 128.9 μg / mL and 94.8 μg / mL, respectively ( Figure 19 ), 106.5 μg / mL and 58.9 μg / mL ( Figure 20 ), 89.0 μg / mL and 102.7 μg / mL ( Figure 21 ), 170.6 μg / mL and 60.0 μg / mL ( Figure 22 The results of cytotoxicity experiments showed that SOFs and POPs had high cytotoxicity and showed no cytotoxicity within the concentration range with the highest antagonistic rate.

[0151] (3) Acute toxicity test

[0152] The present invention uses ICR mice (17-24g) to carry out acute toxicity experiments, with 10-16 mice per group, half male and half female. Taking SOF1 and POP1 as examples, a single injection of tail vein was adopted, with a slow push injection (90s) and an injection volume of 0.1-0.2mL. A normal saline blank control group was set, and the dosage of SOF1 was set to 3.3, 15.7, 31.3, 62.6, 71.2, 78.3, 85.4, 93.9, 99.6, 106.8 and 113.9 mg / kg (Table 2), and the dosage of POP1 was set to 2.6, 5.2, 10.4, 15.6, 16.9, 18.2, 19.5, 20.8 and 26 mg / kg (Table 3). After injection, observation was carried out for a period of 15 days, and weight changes and mortality were recorded. The maximum tolerated dose and median lethal dose (LD50) were fitted using a probit regression model.

[0153] The observation period was extended to 15 days for the control group (SOF1 concentrations of 3.3, 15.7, 31.3, and 62.6 mg / kg, POP1 concentrations of 2.6, 5.2, and 10.4 mg / kg, and a saline blank control group). After 15 days, physiological dissections were performed, and organ indices were measured and recorded. Histopathological changes or abnormalities were observed after hematoxylin-eosin staining. Blood was collected and centrifuged (4°C, 10 minutes, 1200 g) to collect serum. Serum urea nitrogen and alanine aminotransferase levels were measured and recorded to assess renal and liver pathology.

[0154] As shown in Tables 2 and 3, mortality rates for SOF1 and POP1 at different doses were recorded. Probit regression analysis revealed maximum tolerated doses (MTDs) of 71.2 and 15.6 mg / kg for SOF1 and POP1, respectively, with median lethal doses (LD50s) of 92.5 and 20.3 mg / kg, respectively. Converted to a 60 kg adult, these MTDs were 641.5 and 140.6 mg / kg, respectively, with median lethal doses (LD50s) of 833.4 and 182.5 mg / kg, respectively. The therapeutic index (TI) for SOF1 and POP1 was calculated to be >28 and >7.9, respectively, based on the effective dose ([SOF1] = 3.3 mg / mL, [POP1] = 2.6 mg / mL, achieving >90% efficacy) and the median lethal dose (50% lethality), significantly exceeding the required TI for a drug (>3). This indicates that the SOFs and POPs of the present invention (taking SOF1 and POP1 as examples) have high biosafety when used as antagonists of unfractionated heparin and low molecular weight heparin.

[0155] Table 2. Acute toxicity doses of SOF1 tolerated by mice

[0156]

[0157] Table 3. Dosage schedule for mouse tolerance to POP1

[0158]

[0159] like Figure 23 As shown, during the 15-day observation period after SOF1 and POP1 injection, the body weight of mice decreased slightly on the second day after injection, and began to grow normally on the third day, just like the saline blank control group ( Figure 23 a, 23d). After 15 days, physiological anatomy revealed that the organ indices (heart, liver, spleen, lung, and kidney) at different injection doses were all within the normal range and showed no significant difference compared with the saline blank control group ( Figure 23 b, 23e). As shown in the figure, the levels of blood urea nitrogen, which reflects kidney disease, and alanine aminotransferase, which reflects liver disease, were both within the normal range and showed no significant difference compared with the normal saline blank control group ( Figure 23 c, 23f). Figure 24 、 25 The physiological section results of the organs shown in the figure show that SOF1 (3.3-62.6 mg / kg) and POP1 (2.6-10.4 mg / kg) at concentrations higher than the used doses will not cause obvious tissue lesions in the main internal organs of mice. This also shows that SOF1 and POP1 are highly safe at the used doses (3.3 mg / kg and 2.6 mg / kg, respectively).

[0160] This shows that when the SOFs and POPs of the present invention (taking SOF1 and POP1 as examples) antagonize unfractionated heparin and low molecular weight heparin in vivo, they have no obvious toxicity to major organs at effective concentrations (3.3 mg / kg and 2.6 mg / kg) and concentrations higher than the effective concentrations (62.6 mg / kg and 15.6 mg / kg), and have high biosafety.

[0161] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of cationic porous organic polymer SOFs and POPs, characterized in that: Antagonistic drug compositions for preparing heparin anticoagulant drugs; The porous organic polymer SOFs are formed by self-assembly of tetrahedral molecules A1 or A2 and cucurbit[8]uril (CB[8]) main molecules in aqueous phase: The porous organic polymer POPs is formed by crosslinking tetrahedral molecules selected from the group consisting of B1, B2, B3, and B4 through intermolecular [2+2] olefin dimerization cycloaddition reaction. Wherein, the heparin anticoagulant drug is selected from the following group: unfractionated heparin, dalteparin sodium, enoxaparin sodium, nadroparin calcium, or a combination thereof.

2. The use according to claim 1, characterized in that The porous organic polymer SOFs and POPs are three-dimensional structures in a diamond configuration constructed based on tetrahedral molecules.

3. The use according to claim 1, characterized in that The molecular weight of the porous organic polymer SOFs is 230-269 kD.

4. The use according to claim 1, wherein The molecular weight of the porous organic polymer POPs is 64-74 kD.

5. The use according to claim 1, characterized in that The cucurbit[8]uril (CB[8]) has the structure shown in the following formula:

6. Use of a composition of cationic porous organic polymer SOFs, POPs and protamine, characterized in that: Antagonists for the preparation of heparin-type anticoagulants; The porous organic polymer SOFs are formed by self-assembly of tetrahedral molecules A1 or A2 and cucurbit[8]uril (CB[8]) main molecules in aqueous phase: The porous organic polymer POPs is formed by crosslinking tetrahedral molecules selected from the group consisting of B1, B2, B3, and B4 through intermolecular [2+2] olefin dimerization cycloaddition reaction. Wherein, the heparin anticoagulant drug is selected from the following group: unfractionated heparin, dalteparin sodium, enoxaparin sodium, nadroparin calcium, or a combination thereof.

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