Oligomeric cyclodextrin and application thereof in antidote

Abstract

The invention relates to the field of biological medicine, in particular to oligomeric cyclodextrin and application thereof in an antidote. The oligomeric cyclodextrin is prepared from the following raw materials: beta-cyclodextrin and a cross-linking agent; the molar ratio of the beta-cyclodextrin to the cross-linking agent is 1: (8-30); the cross-linking agent is epoxy chloropropane. The oligomeric cyclodextrin is prepared through reaction of a cross-linking agent epichlorohydrin and beta-cyclodextrin, has the characteristics of a monomer cyclodextrin molecule and also has the characteristics of a polymer, a drug can be separated from a binding site on an FXa factor through the inclusion effect of a hydrophobic cavity, the anticoagulant effect of the drug can be reversed, and the drug can be used for preparing the FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa-FXa. Therefore, when the rivaroxaban is excessively used, the blood coagulation function can be prompted to recover to normal in time. The oligomeric cyclodextrin disclosed by the invention has a reversing effect on various oral anticoagulants, is simple in synthetic route, low in raw material cost and relatively good in biological safety when being used under a large dosage, and can fill the blank that the oral anticoagulant is short of a corresponding antidote.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an oligomeric cyclodextrin and application thereof in an antidote. Background Art

[0002] Anticoagulants are a class of drugs used to treat or prevent bleeding disorders. They work by interfering with the normal coagulation process. They can also be used clinically to prevent and treat venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE). Currently, anticoagulants used clinically can be classified into direct thrombin inhibitors and indirect thrombin inhibitors according to their mechanism of action. Indirect thrombin inhibitors include vitamin K antagonists, heparin, etc. Direct coagulants (such as dabigatran and rivaroxaban) can directly bind to the catalytic site of thrombin, quickly and reversibly bind to the clot and dissolve thrombin, or selectively block the active site of factor Xa to exert an anticoagulant effect. However, the use of these anticoagulants will inevitably bring the risk of bleeding, which may cause bleeding complications and may even be life-threatening, and there are potential hidden dangers in some special cases.

[0003] Traditional anticoagulants have an antidote-reversal target therapeutic effect. Vitamin K can be used to reverse warfarin overdose, but too fast intravenous injection of vitamin K may cause chest tightness, bronchial asthma, a sharp drop in blood pressure, allergic reactions, and even life-threatening adverse reactions. Oral vitamin K can also cause gastrointestinal adverse reactions such as nausea and vomiting. Warfarin has a long half-life (about 72 hours), while the serum half-life of vitamin K is about 6 hours, so vitamin K needs to be used frequently and continuously for a long time. Protamine is usually used for anticoagulation in heparin overdose, but protamine has only a partial reversal effect on low molecular weight heparin and has no detoxification activity on heparin derivatives (such as fondaparinux sodium). The side effects caused by protamine are related to immunity and inflammation, and may cause allergic reactions, systemic hypotension, pulmonary hypertension, bronchoconstriction, and bradycardia.

[0004] At present, some vitamin K-independent oral anticoagulants that are widely used in clinical practice are drugs that directly bind to the target (activated factor II (IIa or thrombin) or activated factor X (Xa) and antagonize its activity. These drugs are widely used in patients who cannot use traditional anticoagulants or have difficulty using traditional anticoagulants. However, among these drugs, only the antidote of dabigatran, idarucizumab, has been approved for reversing its anticoagulant effect, while FXa direct inhibitors such as rivaroxaban and apixaban, which are widely used, currently have no antidote with different characteristics. Therefore, research on antidotes for FXa direct inhibitors is imperative.

[0005] Andexanet alfa is a modified inactivated form of recombinant human coagulation factor Xa that can be used as a reversal agent for direct oral FXa inhibitors. The product is used by patients receiving apixaban or rivaroxaban to help reverse their anticoagulant effects in the event of life-threatening or uncontrolled bleeding. However, Andexanet alfa has a certain risk of thrombosis after treatment, and this type of recombinant protein drug is expensive and has a complex preparation process.

[0006] Joost CM Meijers et al. developed a modified cyclodextrin OKL-1111, which can be used as a potential universal reversal agent for anticoagulants. The researchers added 6-mercaptohexanoic acid (6-MHA) to natural γ-cyclodextrin for group modification to generate γ-cyclodextrin with fully modified "arms". OKL-1111 can restore the inhibitory effect of rivaroxaban and apixaban on thrombin and restore thrombin generation in vitro. In vivo, its reversal effect on rivaroxaban and apixaban was evaluated by the mouse tail-cut model and the rabbit Wessler venous stasis model. The results showed that the administration of oral anticoagulants such as rivaroxaban and apixaban increased the induced bleeding time by 3 to 4 times in this model, and OKL-1111 reversed this effect by up to 90%. Genmin Lu et al. designed a recombinant protein r-Antidote as a universal reversal agent for direct oral FXa inhibitors such as rivaroxaban and apixaban. R-Antidote is a truncated form of enzymatically inactive FXa that can bind to direct FXa inhibitors with an affinity comparable to that of native FXa. The researchers determined the potency (Kd) of r-Antidote binding to three direct FXa inhibitors (betrixaban, rivaroxaban, and apixaban) and compared them with inhibition of purified human FXa using peptide substrates, finding that it dose-dependently reversed the inhibitory activity of small molecule FXa inhibitors, but did not change the rate of Xa cleavage of the peptide substrate in the absence of inhibitors. r-Antidote reversed the anticoagulant effect of rivaroxaban in human plasma through in vitro coagulation tests, and further studied the function of r-Antidote in restoring hemostasis in rat tail amputation models and rabbit liver laceration rabbit models. The results showed that r-Antidote could significantly reverse the increased bleeding volume and prolonged bleeding time caused by rivaroxaban, proving that it has the effect of reversing rivaroxaban. However, the cost of this technology is high, and the use of recombinant proteins may cause side effects such as allergies, making it difficult to be widely used.

[0007] Under such circumstances, there is an urgent need to provide an antidote with a simple preparation process, low cost, good reversal effect and no side effects. Summary of the invention

[0008] Since the molecules of FXa direct coagulation inhibitors such as rivaroxaban and apixaban are relatively small and highly hydrophobic, they can be included with cyclodextrin, thereby dissociating them from the FXa binding site and reversing their anticoagulant effect. However, the spontaneous inclusion binding force of the existing common cyclodextrin and inhibitors such as rivaroxaban is relatively weak, and can only play a certain solubilizing effect, and it is impossible to completely include them and block their effects. The present invention prepares a kind of oligomeric cyclodextrin by reacting the cross-linking agent epichlorohydrin with beta-cyclodextrin, which has the characteristics of monomeric cyclodextrin molecules (such as hollow cage structures and the ability to interact with the interior of the molecule), and also has the characteristics of polymers (such as high molecular weight and chain structures), and can separate the drug from the binding site on the FXa factor by the inclusion action of the hydrophobic cavity, and reverse its anticoagulant effect, so that when rivaroxaban appears to be overdosed, it can promptly promote the recovery of coagulation function to normal.

[0009] The first aspect of the present invention provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include cyclodextrin and a cross-linking agent; the molar ratio of the cyclodextrin to the cross-linking agent is 1:(8-30); it can be listed as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30.

[0010] More preferably, the molar ratio of the cyclodextrin to the cross-linking agent is 1:(8-25); the prepared oligomeric cyclodextrin has strong safety.

[0011] Examples of the cyclodextrin include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0012] Preferably, the cyclodextrin is β-cyclodextrin Preferably, the cross-linking agent is epichlorohydrin.

[0013] The present invention regulates the cross-linking degree and molecular weight of oligomeric cyclodextrin by exploring the feed ratio of epichlorohydrin to β-cyclodextrin. After a large number of experimental studies, it is found that when the molar ratio of β-cyclodextrin to epichlorohydrin is 1: (8-30), the obtained oligomeric cyclodextrin has a stronger reversal effect on rivaroxaban.

[0014] In some preferred embodiments, the preparation steps of the oligomeric cyclodextrin include: adding β-cyclodextrin to an alkaline solution, heating to 55-65°C, adding a cross-linking agent dropwise, reacting for 1.5-2.5 hours to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, and then performing membrane dialysis and drying to obtain an oligomeric cyclodextrin product.

[0015] The reaction equation is as follows. Under alkaline conditions, the hydroxyl group (-OH) of cyclodextrin reacts with the epoxy group of epichlorohydrin to form new chemical bonds, which are further subjected to cross-linking, self-polymerization and hydrolysis to generate oligomeric cyclodextrin. The oligomeric cyclodextrin obtained has both the characteristics of monomeric cyclodextrin molecules (such as hollow cage structure and the ability to interact with the interior of the molecule) and polymer characteristics (such as high molecular weight and chain structure).

[0016] The alkaline solution may be exemplified by aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous calcium hydroxide solution, aqueous ammonia, and the like.

[0017] Preferably, the alkaline solution is an aqueous sodium hydroxide solution.

[0018] More preferably, the concentration of the sodium hydroxide aqueous solution is 100-200 g / L.

[0019] More preferably, the preparation steps of the oligomeric cyclodextrin include: adding β-cyclodextrin to an alkaline solution, heating to 60°C, adding a cross-linking agent, reacting for 2 hours to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, and then performing membrane dialysis and drying to obtain an oligomeric cyclodextrin product.

[0020] Preferably, the molecular weight of the filter membrane for membrane dialysis is 15-25 kDa.

[0021] More preferably, the molecular weight of the filter membrane for membrane dialysis is 20 kDa.

[0022] Preferably, the pH is adjusted to neutral. More preferably, the pH is adjusted to neutral using aqueous hydrochloric acid.

[0023] The drying method may include vacuum drying, oven drying, freeze drying, etc.; freeze drying is preferred.

[0024] The second aspect of the present invention provides an application of an oligomeric cyclodextrin, wherein the oligomeric cyclodextrin is applied as an antidote.

[0025] Note: An antidote is a substance that can interact with a specific drug (such as the anticoagulant rivaroxaban) to reduce or eliminate the pharmacological effect of the drug (such as anticoagulant effect), thereby reversing its biological activity or toxicity.

[0026] In some preferred embodiments, the reversal target of the antidote is rivaroxaban.

[0027] Cyclodextrin and its derivatives can be used as carriers, stabilizers or modifiers to improve the solubility, stability or targeting of drugs. It is worth noting that cyclodextrin can be used as an auxiliary in coagulation scenarios. For example, if coagulation function needs to be restored during surgery, the patient can be injected with cyclodextrin-encapsulated hemostatic drugs to restore coagulation. The purpose is to deal with the problem of emergency postoperative hemorrhage and inability to coagulate. The present invention unexpectedly discovered that the use of oligomeric cyclodextrin can spontaneously encapsulate rivaroxaban (an anticoagulant drug that causes inability to coagulate and is mostly used by patients with thrombosis) through hydrophobic cavities, and the cross-linked polymer network allows rivaroxaban to be firmly bound to oligomeric cyclodextrin, dissociating it from the FXa site so that it can be quickly cleared from the blood, thereby promoting the recovery of coagulation function.

[0028] In some preferred embodiments, the mass ratio of the oligomeric cyclodextrin to rivaroxaban is 1:(4-20).

[0029] In some preferred embodiments, when the oligomeric cyclodextrin is used as an antidote, the administration method is injection; specifically, the oligomeric cyclodextrin and the prepared physiological saline are mixed into a mixed solution, and then injected. More preferably, the concentration of the oligomeric cyclodextrin in the mixed solution is 0.5-2 mg / kg, and more preferably 1 mg / kg.

[0030] Beneficial effects: The present invention provides an oligomeric cyclodextrin and its application, which has the following advantages: (1) The present invention generates cross-linked oligomeric cyclodextrin by reacting epichlorohydrin with β-cyclodextrin, which can maintain the inherent macrocyclic structure and functional hydroxyl groups of cyclodextrin, and has the properties of a polymer, showing a high degree of "integration" and "synergy" effects. Oligomeric cyclodextrin has the advantages of both cross-linked polymers and cyclodextrins, and also gives cross-linked cyclodextrin special physical and chemical properties.

[0031] (2) The present invention uses a self-made oligomeric cyclodextrin to reverse the anticoagulant rivaroxaban and exert a procoagulant effect. The oligomeric cyclodextrin spontaneously includes rivaroxaban through the hydrophobic cavity, and the cross-linked polymer network allows rivaroxaban to be firmly bound to the oligomeric cyclodextrin, dissociating it from the FXa site, allowing it to be quickly cleared from the blood, and exerting the function of promoting the recovery of coagulation function.

[0032] (3) The present invention uses the mature excipient β-cyclodextrin as the raw material. The synthesized oligomeric cyclodextrin does not have the side effects such as allergies that may be caused by recombinant protein drugs, has high biocompatibility, and can maintain a high level of safety even when administered in large doses; it is easy to implement.

[0033] (4) The present invention has a simple synthesis route, a convenient preparation process, low raw material prices, and extremely high economic application value.

[0034] (5) The present invention fills the gap of the current lack of corresponding antidotes for oral anticoagulants and has huge market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 . Schematic diagram of the reaction of oligomeric cyclodextrin; Figure 1 (a) is the cross-linking process, (b) is the self-aggregation process, and (c) is the degradation process; Figure 2 .Comparison chart of in vitro coagulation test results; Figure 2 A to G in the table correspond to different oligomeric cyclodextrins, A corresponds to Example 1, B corresponds to Example 2, C corresponds to Example 3, D corresponds to Example 4, E corresponds to Example 5, F corresponds to Example 6, and G corresponds to Example 2; information on samples a to j is shown in Table 1; Figure 3 .Coagulation index test result chart; Figure 3 a~l in the figure correspond to different cyclodextrins, a corresponds to Example 1, b corresponds to Example 2, c corresponds to Example 4, d corresponds to Example 5, e corresponds to Example 6, f corresponds to Example 7, g corresponds to Example 1, h corresponds to Example 2, i corresponds to Example 1, j corresponds to GAMA-CD, k corresponds to SBE-γ-CD, and l corresponds to γ-CD; wherein the triangles in the figure represent experimental data points; Figure 4 .RIV coagulation index test results; "**" means p<0.01, "****" means p<0.0001; Figure 5 .Thromboelastometry test results; Figure 5 a is RWB, b is RWB+RIV, c is RWB+RIV+100μM CCD, and D is RWB+RIV+200μM CCD; Figure 6 . Comparison of coagulation time; “*” indicates p<0.1, “**” indicates p<0.01; Figure 7 .Comparison chart of coagulation comprehensive index; "**" indicates p<0.01, "***" indicates p<0.001; Figure 8 .Comparison of fibrin function; "*" indicates p<0.1, "**" indicates p<0.01; Fig. 9 . 1 H-NMR test results; Fig. 9A1 is CCD, A2 is RIV, A3 is RIV:CCD=20:1; B1 is ECH:β-CD=0.1:1, B2 is ECH:β-CD=1:1, B3 is ECH:β-CD=4:1, B4 is ECH:β-CD=8:1, B5 is ECH:β-CD=15:1, B6 is ECH:β-CD=30:1; each ratio is a molar mass ratio; Fig.10 .ITC measurement result diagram; the dots in the diagram represent experimental data points; Fig.11 .The results of prothrombin time measurement in BALB / c mice; Fig.11 a in the figure is the PT measurement result after intragastric administration of rivaroxaban. The black line in the figure is the control group, the red line is the rivaroxaban group, and the green, purple, pink and blue lines are all rivaroxaban + oligomeric cyclodextrin groups, in which the CCD concentrations are 0.5g / kg, 1.0g / kg, 1.5g / kg and 2.0g / kg respectively; Fig.11 b in the figure is the PT measurement result after CCD injection; Fig.11 c in it is the coagulation index after injection of CCD; Fig.11 The triangle in a represents the experimental data points. Fig.11 The dots, squares, triangles, inverted triangles, and diamonds in b represent experimental data points. Fig.11 The triangle in c represents the experimental data point; "*" represents p < 0.1, "**" represents p < 0.01, "***" represents p < 0.001, and "****" represents p < 0.0001; Fig.12 .Results of tail bleeding experiment on BALB / c mice; dots, squares, triangles, inverted triangles, and diamonds represent experimental data points; "****" indicates p<0.0001; Fig.13 .Results of liver bleeding experiment in BALB / c mice; “*” indicates p<0.1, “****” indicates p<0.0001; Fig.14 .BALB / c mouse growth curve; dots, squares, triangles, inverted triangles, and diamonds represent experimental data points; Fig.15 .BALB / c mouse blood routine test results; dots, squares, triangles, inverted triangles, and diamonds represent experimental data points; Fig.16 .BALB / c mouse blood biochemistry results; dots, squares, triangles, inverted triangles, and diamonds represent experimental data points; Fig.17.Results of organ index of BALB / c mice; dots, squares, triangles, inverted triangles, and diamonds represent experimental data points; Fig.18 .Results of organ optical microscopy observation; each column in the figure corresponds to the same organ, and the five columns from left to right are heart, liver, spleen, lung, and kidney; the scale bar in the figure is 100μm; Fig.19 .Hemolysis test results; Fig.19 A to F in the figure correspond to different oligomeric cyclodextrins, A corresponds to Example 1, B corresponds to Example 2, C corresponds to Example 4, D corresponds to Example 5, E corresponds to Example 6, and F corresponds to Example 7. DETAILED DESCRIPTION

[0036] The reaction diagram of preparing oligomeric cyclodextrin in the embodiment of the present invention is shown in Figure 1 .

[0037] Note: Unless otherwise specified, the solvents of the solutions involved in the present invention are all water; and the raw materials used are all commercially available.

[0038] Example Example 1 This embodiment provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin (β-CD) and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:4.

[0039] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 2.75mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral using a 5wt% HCl aqueous solution, then dialyzing using a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0040] Example 2 This embodiment provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:8.

[0041] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 5.5mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral using a 5% HCl aqueous solution, then dialyzing using a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0042] Example 3 This embodiment provides an oligomeric cyclodextrin, wherein raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:10.

[0043] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 6.88mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral using a 5% HCl aqueous solution, then dialyzing using a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0044] Example 4 This embodiment provides an oligomeric cyclodextrin, wherein raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:15.

[0045] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 10.31mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5% HCl aqueous solution, then dialyzing with a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0046] Example 5 This embodiment provides an oligomeric cyclodextrin, wherein raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:20.

[0047] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 13.75mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5% HCl aqueous solution, then dialyzing with a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0048] Example 6 This embodiment provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:25.

[0049] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 17.19mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5% HCl aqueous solution, then dialyzing with a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0050] Example 7 This embodiment provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:30.

[0051] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 20.63mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5% HCl aqueous solution, then dialyzing with a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0052] Comparative Example 1 This comparative example provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:0.1.

[0053] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropping 0.07mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5% HCl aqueous solution, then dialyzing with a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0054] Comparative Example 2 This comparative example provides an oligomeric cyclodextrin, wherein the raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:1.

[0055] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52g of sodium hydroxide in 25ml of water, then adding 10g of β-cyclodextrin, heating to 60°C, dropwise adding 0.69mL of epichlorohydrin, reacting for 2h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral using a 5% HCl aqueous solution, then dialyzing using a 20kDa membrane, and freeze-drying to obtain an oligomeric cyclodextrin product.

[0056] Performance Testing The abbreviations of some samples involved in the following tests are explained as follows: CCD: oligomeric cyclodextrin; in tests 1 to 8, the oligomeric cyclodextrin samples used were from Example 2 unless otherwise specified.

[0057] CD: cyclodextrin; β-CD: commercially available cyclodextrin, manufacturer: McLean, CAS number: 7585-39-9; Cationic-CD: cationic cyclodextrin, commercially available cyclodextrin, manufacturer Zhiyuan Biotechnology, batch number: CC20240424P; HP-γ-CD: commercially available cyclodextrin, manufacturer: Zhiyuan Biotechnology, batch number: HG20230709; SBE-γ-CD: commercially available cyclodextrin, manufacturer Zhiyuan Biotechnology, product batch number: SB20240330; RIV: rivaroxaban; ECH: epichlorohydrin.

[0058] RWB: rat whole blood.

[0059] And, unless otherwise specified, the control samples involved in each test are all base control samples.

[0060] 1. In vitro coagulation test Test method: 1) SD rats were anesthetized with isoflurane, and fresh blood samples were collected and added to a 10 mL centrifuge tube containing an anticoagulant (3.8 wt% sodium citrate aqueous solution). The ratio of fresh blood sample to anticoagulant was 9:1 to obtain anticoagulated blood. The anticoagulated blood was stored on ice and the experiment was performed within 4 h.

[0061] 2) Take a 5 ml EP tube and add 98 μL of anticoagulated blood into each tube, and set up ① control group, ② rivaroxaban group, and ③ rivaroxaban + oligomeric cyclodextrin group.

[0062] 3) Add 1 μL DMSO to the control group and mix thoroughly. Add 1 μL 2 mg / mL rivaroxaban solution (solvent DMSO) to the rivaroxaban group and the rivaroxaban + oligocyclodextrin group. The final rivaroxaban concentration in the blood is 20 μg / mL. Mix thoroughly.

[0063] 4) Add 1 μL of saline to the control group and rivaroxaban group, and add 1 μL of oligocyclodextrin solution (solvent is saline) to the rivaroxaban + oligocyclodextrin group. The final concentration of oligocyclodextrin in the blood is 1.25, 2.5, 5, 10, 20, 40, 80, 160 μg / mL. Mix thoroughly and incubate in a 37°C constant temperature incubator for 10 min.

[0064] 5) After the incubation, add 20 μL of 0.2M calcium chloride solution (solvent is water) to activate the blood and start coagulation. Mix thoroughly again and incubate in a 37℃ constant temperature incubator for 10 minutes. Observe the coagulation effect. Figure 2 . Figure 2 The information of samples a~i is shown in Table 1. Then 3 mL of deionized water was added along the side wall, and the mixture was incubated for 5 min to completely rupture the blood cells in the unsolidified blood clots and release hemoglobin, and then the absorbance was measured at 540 nm. The above groups were repeated 3 times. Figure 3 A~G in the table correspond to different oligomeric cyclodextrins. Figure 3 Among them, A corresponds to Example 1, B corresponds to Example 2, C corresponds to Example 3, D corresponds to Example 4, E corresponds to Example 5, F corresponds to Example 6, and G corresponds to Example 2.

[0065] The hemoglobin content of the ruptured uncoagulated blood was detected at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (BioTek®, Synergy H1) and the coagulation index was calculated. The results are shown in Figure 3 . Figure 3 Ctrl corresponds to sample a (control group), RIV corresponds to sample b (rivaroxaban group), and 1.25~160 represents the concentration of oligomeric cyclodextrin in the rivaroxaban + oligomeric cyclodextrin group (unit: μg / mL). Figure 3 Samples a~j in each group of tests correspond to different cyclodextrins, a corresponds to Example 1, b corresponds to Example 2, c corresponds to Example 4, d corresponds to Example 5, e corresponds to Example 6, f corresponds to Example 7, g corresponds to Example 1, h corresponds to Example 2, i corresponds to Example 1 (RIV 4μg / mL), j corresponds to GAMA-CD, k corresponds to SBE-γ-CD, and l corresponds to γ-CD.

[0066] At the same time, the coagulation index of RIV was measured, and the results are shown in Figure 4 .

[0067] Table 1

[0068] Note: In Table 1, - represents no addition, and + represents addition.

[0069] from Figure 2 It can be seen that the addition of oligomeric cyclodextrin solutions with a concentration range of 1.25-80 μg / mL can reverse the effects of rivaroxaban in whole blood ex vivo and restore blood coagulation function. Figure 3 , Figure 4 The results showed that compared with the rivaroxaban group, the hemoglobin content in the rivaroxaban + oligomeric cyclodextrin group decreased, the coagulation index decreased, and the coagulation effect was restored.

[0070] 2. Rotational thromboelastography (ROTEM) experiment SD rats were anesthetized with isoflurane, and the rat ex vivo blood was collected. 1 mL of 3.2% sodium citrate aqueous solution was added to 9 mL of ex vivo blood sample (RWB). 980 μL of whole blood was mixed with 10 μL of anticoagulant rivaroxaban solution (solvent DMSO). The concentration of rivaroxaban in the blood was 50 μM, and the mixture was incubated at 37°C for 5 minutes. The reversal agent oligomeric cyclodextrin dissolved in normal saline was added to make its concentration in the blood 100 μM or 200 μM, and the mixture was incubated at 37°C for 10 minutes to obtain a blood-antidote suspension. 1.0 mL of the blood-antidote suspension was transferred to an automatic thromboelastography instrument (LEPU, LEPU-Auto8) for measurement. The measurement results are shown in Figures 5 to 8 ; At the same time, set the control sample contral. Figure 5 a in the figure is the RWB group, b is the RWB+RIV group, c is the RWB+RIV+100μM CCD group, and d is the RWB+RIV+200μM CCD group; in the thromboelastogram, the abscissa is the time of coagulation, and the ordinate is the amplitude. Figure 6 The intermediate coagulation time (R) refers to the time from the initiation of the coagulation system to the beginning of fibrin clot formation (TEG tracing amplitude reaches 2 mm); Figure 7 The coagulation index (CI) is an indicator that comprehensively reflects the coagulation function of the sample. It is a comprehensive result based on the prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen level. These indicators jointly reflect the activity of coagulation factors and the fibrin formation process in the blood; the fibrin function (angle) refers to the angle between the horizontal line and the tangent line from the blood clot formation point to the maximum arc of the curve.

[0071] 3. 1 H-NMR test Since rivaroxaban is soluble in D6-DMSO (dimethyl sulfoxide) and has poor solubility in other deuterated reagents, rivaroxaban, oligocyclodextrin and rivaroxaban + oligocyclodextrin were dissolved in D6-DMSO, mixed, and stirred in a magnetic stirrer at room temperature for 1 h at a stirring speed of 500 rpm. A 400M nuclear magnetic resonance spectrometer (BRUKER®, Avance III) was used for the analysis. 1 H-NMR determination, the data were processed with Mestrenova®, and the chemical shifts of the characteristic peaks of rivaroxaban in the mixture of rivaroxaban + oligomeric cyclodextrin were compared with those of the characteristic peaks of rivaroxaban solution, with the D6-DMSO solvent peak (2.5000ppm) as reference. 1 The changes and shifts of the characteristic peaks of rivaroxaban in H-NMR determined whether rivaroxaban and oligomeric cyclodextrin spontaneously underwent inclusion complexation. The test results are shown in Table 2 and Fig. 9The results of A1~A3 in the above table confirm that rivaroxaban spontaneously undergoes inclusion complexation with oligomeric cyclodextrin. The mass spectrometry results of rivaroxaban and β-CD mixtures at different concentrations were also measured. Fig. 9 B1~B6 in the.

[0072] Table 2

[0073] 4. Isothermal Titration Calorimeter (ITC) Experiment The in vitro affinity of oligomeric cyclodextrin to rivaroxaban can be determined by isothermal titration calorimetry (MicroCal®, PEAQ-ITC). A 2 mM oligomeric cyclodextrin solution (solvent is PBS buffer containing 5% DMSO) is added to a 100 uM rivaroxaban sample pool (solvent is PBS buffer containing 5% DMSO) via a syringe. 27 injections are made. After the titration, the MicroCal® PEAQ-ITC Analysis Software is used for analysis. The results are shown in Fig.10 The in vitro binding affinity (Ks) of oligocyclodextrin to rivaroxaban was determined to be 5000M -1 The enthalpy change (△H) was -2.18KJ / mol, indicating that oligomeric cyclodextrin had good in vitro affinity with rivaroxaban.

[0074] 5. Prothrombin time (PT) experiment in BALB / c mice 5.1 Rivaroxaban + oligomeric cyclodextrin PT determination 1) Experimental animals: 218 male BALB / c mice.

[0075] 2) Prepare reagents: 20 mg / kg rivaroxaban solution (the solvent is a mixture of PEG400, anhydrous ethanol and water in a volume ratio of 3:1:1, and the administration volume for each mouse is 0.05 mL / 10 g); 0.5 g / kg, 1.0 g / kg, 1.5 g / kg and 2 g / kg oligomeric cyclodextrin solution (the solvent was normal saline, and the dosing volume for each mouse was 0.05 mL / 10 g).

[0076] 3) Experimental groups: Blank group: 8 Rivaroxaban model group: 50 rats, 10 rats at 1 h, 1h10min, 2.5h, 3.5h and 5h Rivaroxaban + 0.5g / kg oligocyclodextrin group: 40 mice, 10 mice each at 1h10min, 2.5h, 3.5h and 5h Rivaroxaban + 1.0g / kg oligomeric cyclodextrin group: 40 mice, 10 mice each at 1h10min, 2.5h, 3.5h and 5h Rivaroxaban + 1.5g / kg oligomeric cyclodextrin group: 40 mice, 10 mice each at 1h10min, 2.5h, 3.5h and 5h Rivaroxaban + 2.0g / kg oligomeric cyclodextrin group: 40 mice, 10 mice each at 1h10min, 2.5h, 3.5h and 5h Note: The recorded time of each group was calculated from the oral administration of rivaroxaban, that is, 1h represented one hour after the oral administration of rivaroxaban; oligomeric cyclodextrin was injected 1h after the oral administration of rivaroxaban, and blood was collected 1.5h, 2.5h and 4h after the injection of oligomeric cyclodextrin, thus corresponding to the 2.5h, 3.5h and 5h groups, respectively.

[0077] 4) Experimental process: Step 1: Before intragastric administration of rivaroxaban, blood was collected from the mouse eyeballs to obtain the PT (s) of the mice in a normal state. Ten mice were treated.

[0078] Step 2: Both the rivaroxaban group and the rivaroxaban + oligomeric cyclodextrin group were orally administered 20 mg / kg of rivaroxaban solution by gavage.

[0079] Step 3: One hour after oral administration, 10 mice in the rivaroxaban model group were given eye blood samples to measure PT. At the same time, 160 mice (40 mice in each group) in the rivaroxaban + oligomeric cyclodextrin group were injected with oligomeric cyclodextrin solution via tail vein according to the concentrations of oligomeric cyclodextrin: 0.5 g / kg group (1h10min, 2.5h, 3.5h and 5h group), 1.0 g / kg group (1h10min, 2.5h, 3.5h and 5h group), 1.5 g / kg group (1h10min, 2.5h, 3.5h and 5h group), and 2.0 g / kg group (1h10min, 2.5h, 3.5h and 5h group).

[0080] Step 4: In each group, mice were anesthetized with isoflurane at different time points, their whiskers were cut, and their eyes were enucleated for blood collection. Blood was collected from the eyes of mice and the supernatant was immediately centrifuged at 3000 rpm and 4°C to obtain plasma for the determination of PT.

[0081] 5) Specific methods for PT determination: Collect mouse eyeball blood using an anticoagulant tube, gently mix the EP tube by inverting it upside down, and then immediately put it in ice. Keep it in an ice bath for less than 4 hours. Centrifuge it at 3000 r / min for 15 minutes within 4 hours, and then use a pipette to draw the supernatant plasma. Use a prothrombin (PT) detection kit (Yuanye Biotechnology®, R24682-100T) to detect and record the sample PT value according to the method of the PT kit. The results are shown in Fig.11 a in.

[0082] like Fig.11 As shown in a in the figure, after oral gavage of rivaroxaban, the PT of mice was significantly prolonged. When the corresponding dose of oligomeric cyclodextrin was injected at the time point when rivaroxaban had the most obvious effect, the PT of mice decreased, and as time went on and the action time increased, it was able to return to a near normal level, reflecting that the oligomeric cyclodextrin of the present invention has a significant reversal effect.

[0083] 5.2 Oligomeric cyclodextrin PT determination The experimental method was the same as in 5.1. The test was performed without intragastric administration of rivaroxaban. Instead, different concentrations of oligo-cyclodextrin (CCD) were injected 1.5 h later (corresponding to Fig.11 PT was measured at 2.5h on the horizontal axis of a in the figure. The results are shown in Fig.11 In b, the results show that direct injection of oligomeric cyclodextrin does not have a significant effect on PT. At the same time, the coagulation time of injection of different concentrations of oligomeric cyclodextrin was measured, and the results are shown in Fig.11 In center c, the results show that oligomeric cyclodextrin (CCD) alone cannot restore normal coagulation.

[0084] 6. BALB / c mouse tail bleeding liver bleeding model experiment 1) Experimental animals: 220 male BALB / c mice.

[0085] 2) Experimental reagents: 20 mg / kg rivaroxaban solution (the solvent is PEG400, anhydrous ethanol and water mixed in a mass or volume ratio of 3:1:1, and the administration volume for each rat is 0.05 mL / 10 g); 0.5 g / kg, 1.0 g / kg, 1.5 g / kg and 2 g / kg oligomeric cyclodextrin solution (the solvent was normal saline, and the dosing volume for each mouse was 0.05 mL / 10 g).

[0086] 3) Experimental groups: Blank group: 8 Rivaroxaban model group Rivaroxaban + 0.5g / kg oligomeric cyclodextrin group Rivaroxaban + 1.0g / kg oligomeric cyclodextrin group Rivaroxaban + 1.5g / kg oligomeric cyclodextrin group Rivaroxaban + 2.0g / kg oligomeric cyclodextrin group 4) Experimental process: Step 1: The mice in the blank group were intragastrically administered with normal saline (dosing volume 0.05 mL / 10 g), and the mice in the rivaroxaban model group and the rivaroxaban + oligomeric cyclodextrin group were intragastrically administered with rivaroxaban solution (dosing volume 0.05 ml / 10 g).

[0087] Step 2: Use a vernier caliper to measure the average tail diameter of the mouse at 2 cm from the tail to obtain the average diameter D.

[0088] Step 3: Use isoflurane to anesthetize the mouse. 60 minutes after gavage with rivaroxaban, use a scalpel to cut off the mouse's tail at the length of the diameter D. Add 1 mL of water to a 2 mL EP tube, place the EP tube at the tail of the cut-tailed mouse, and extend the wound below the liquid surface to collect blood. Collect blood for 30 minutes, dilute 10 times, and use an enzyme reader to measure the absorbance at 450 nm to determine the amount of tail bleeding (the higher the absorbance, the higher the surface bleeding). Test results are shown in Fig.12 .

[0089] Step 4: Liver bleeding: 10 minutes after collecting tail blood, immediately spread the abdomen with surgical scissors and make a 1 cm long and 3 mm deep wound on the left liver lobe of the rat. Weigh the filter paper and cotton balls in advance and place the filter paper under the liver lobe. Collect blood every 4 seconds with clean cotton balls. Weigh the filter paper and cotton balls to determine the blood loss in 15 minutes. Test results are shown in Fig.13 .

[0090] Fig.12 , 13 As shown, after oral gavage of rivaroxaban, the amount of liver bleeding and tail bleeding in mice increased significantly. When a corresponding dose of oligomeric cyclodextrin was injected at the time point when rivaroxaban had a significant effect, the amount of liver bleeding and tail bleeding in mice decreased and were able to be restored to near normal levels, indicating that the oligomeric cyclodextrin of the present invention has a significant reversal effect.

[0091] 7. Safety evaluation of oligomeric cyclodextrins Male BALB / C mice weighing 24-28g were divided into groups. The control group was intravenously injected with normal saline, and the mice in the drug-treated group were intravenously injected with oligomeric cyclodextrin solution (solvent was normal saline). Their body weight was measured every day, and their general behavior was observed. The monitoring was continued for 21 days, and the mice were euthanized after 21 days. Blood samples were collected for hematological analysis of whole blood cells, red blood cells, platelets, hemoglobin, etc. The concentrations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and urea (UREA) were determined using a kit. The main organs of the mice were separated, weighed, and the organ index was calculated (the organ index is the ratio of the organ weight to the body weight of each mouse). The test results are shown in the table below. Fig.14 , Fig.15 , Fig.16 , Fig.17 At the same time, H&E staining (hematoxylin-eosin staining) was performed on the heart, liver, spleen, lung, and kidney tissue sections of the mice, and the imaging results were obtained using the integrated cell imaging analysis system (EVOSM7000). Fig.18 , Fig.18 Each column in the figure corresponds to the same organ, and the five columns from left to right are heart, liver, spleen, lung, and kidney; the scale bar in the figure is 100 μm. The experimental results show that the oligomeric cyclodextrin prepared by the present invention has good safety at an addition amount of 0.5-2.0 g / kg.

[0092] 8. Hemolysis Experiment 1) Take blood from a SD rat and collect it in an anticoagulant tube containing an anticoagulant (3.8% sodium citrate aqueous solution). The ratio of fresh blood sample to anticoagulant is 9:1. Centrifuge at 3000 rpm and 4°C for 15 minutes to remove the supernatant plasma and platelets and obtain red blood cells.

[0093] 2) Resuspend the red blood cells in saline 3 to 4 times, using the same centrifugation conditions.

[0094] 3) Prepare 2% red blood cell suspension with normal saline (2 mL red blood cells + 98 mL normal saline) 4) Add 500 μL of red blood cell suspension to the EP tube, 500 μL of red blood cell lysate to the positive control, 500 μL of saline to the negative control, and 500 μL of a series of concentration gradient oligocyclodextrin to the experimental group. Mix well and incubate at 37 degrees for 3 hours.

[0095] 5) Centrifuge the sample at 3000 rpm and 4°C for 15 minutes. Take the supernatant and measure it using an ELISA reader at a wavelength of 540 nm.

[0096] 6) Data processing: Hemolysis rate = (sample - negative control) / (positive control - negative control) * 100%; test results are shown in Fig.19 . Fig.19A to F in the figure correspond to different oligomeric cyclodextrins, A corresponds to Example 1, B corresponds to Example 2, C corresponds to Example 4, D corresponds to Example 5, E corresponds to Example 6, and F corresponds to Example 7.

[0097] Note: A hemolysis rate of <5% is safe.

[0098] Combined with the analysis of the experimental results, it can be seen that the oligomeric cyclodextrin prepared by the present invention can effectively restore the whole blood coagulation obstruction caused by rivaroxaban in vitro, and restore the coagulation function to normal. At the same time, it can effectively reverse the phenomena of prolonged thrombin time, decreased fibrin function, decreased comprehensive coagulation index, etc. caused by rivaroxaban, and the blood coagulation function can be restored after adding oligomeric cyclodextrin. And the addition of oligomeric cyclodextrin to mice can restore the prolonged prothrombin time (PT) of mouse blood caused by rivaroxaban. The evaluation results of the mouse tail-cut model and the mouse liver bleeding model showed that the administration of oral anticoagulants such as rivaroxaban induced an increase in bleeding time in the model, and this effect could be reversed after the tail vein injection of oligomeric cyclodextrin. The safety evaluation results showed that oligomeric cyclodextrin did not affect the weight gain of animals in vivo, and the tissue staining section evaluation and blood routine blood biochemical test showed that it had no toxicity to the heart, liver, spleen, lungs and kidneys. The oligomeric cyclodextrin prepared by the present invention had excellent reversal effect and strong safety.

Claims

1. An oligomeric cyclodextrin, characterized in that: The raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent; the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:(8-30); The cross-linking agent is epichlorohydrin.

2. The oligomeric cyclodextrin according to claim 1, characterized in that The preparation steps of the oligomeric cyclodextrin include: adding β-cyclodextrin to an alkaline solution, heating to 55-65° C., dropping a crosslinking agent, reacting for 1.5-2.5 hours to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, then performing membrane dialysis, and drying to obtain an oligomeric cyclodextrin product.

3. The oligomeric cyclodextrin according to claim 2, characterized in that The preparation steps of the oligomeric cyclodextrin include: adding β-cyclodextrin to a sodium hydroxide aqueous solution, heating to 60° C., dropping a crosslinking agent, reacting for 2 hours to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, and then performing membrane dialysis and drying to obtain an oligomeric cyclodextrin product.

4. The oligomeric cyclodextrin according to claim 2 or 3, characterized in that The molecular weight of the filter membrane for the membrane dialysis is 15-25 kDa.

5. The oligomeric cyclodextrin according to claim 4, characterized in that The molecular weight of the filter membrane of the membrane dialysis is 20 kDa.

6. The oligomeric cyclodextrin according to claim 3, characterized in that The concentration of the sodium hydroxide aqueous solution is 100-200 g / L.

7. The oligomeric cyclodextrin according to claim 2 or 3, characterized in that Adjust pH to neutral.

8. The oligomeric cyclodextrin according to claim 2 or 3, characterized in that The drying is freeze drying.

9. A use of the oligomeric cyclodextrin according to claim 1, characterized in that: The oligomeric cyclodextrin is applied to an antidote, and the reversal object of the antidote is rivaroxaban.

10. The use of oligomeric cyclodextrin according to claim 9, characterized in that: The mass ratio of the oligomeric cyclodextrin to rivaroxaban is 1:(4-20).

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