An oligocyclodextrin and its application in an antidote

Abstract

The present invention relates to the field of biomedicine, and particularly to an oligocyclodextrin and its application in an antidote. The raw materials for preparing the oligocyclodextrin include β-cyclodextrin and a crosslinking agent; the molar ratio of the β-cyclodextrin to the crosslinking agent is 1:(8-30); the crosslinking agent is epichlorohydrin. The present invention prepares an oligocyclodextrin by reacting the crosslinking agent epichlorohydrin with β-cyclodextrin. It has both the characteristics of a monomeric cyclodextrin molecule and the characteristics of a polymer, and can detach the drug from the binding site on the FXa factor through the inclusion effect of the hydrophobic cavity, reversing its anticoagulant effect, so as to promote the recovery of normal blood coagulation function in a timely manner when the use of rivaroxaban is excessive. The oligocyclodextrin of the present invention has a reversing effect on a variety of oral anticoagulants, has a simple synthesis route, low raw material cost, and good biosafety when used in large doses, and can fill the gap in the lack of corresponding antidotes for oral anticoagulants.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an oligocyclodextrin and its application in an antidote. Background Art

[0002] Anticoagulants are a class of drugs used to treat or prevent hemorrhagic diseases, which act by interfering with the normal blood coagulation process. Clinically, they can also be used to prevent and treat venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE). Currently, the 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, rivaroxaban) can directly bind to the catalytic site of thrombin, rapidly and reversibly bind to blood clots and dissolve thrombin, or selectively block the active site of factor Xa to exert anticoagulant effects. However, the use of these anticoagulants inevitably brings the risk of bleeding, which may cause bleeding complications and even endanger life, and there are potential hazards in some special cases.

[0003] Traditional anticoagulants have antidotes to reverse the target therapeutic effect. When the use of warfarin is excessive, vitamin K can be used for reversal. However, rapid intravenous injection of vitamin K may cause adverse reactions such as chest tightness, bronchial asthma, sharp drop in blood pressure, allergic reactions and even endanger life, and oral vitamin K will also cause gastrointestinal adverse reactions such as nausea and vomiting. The half-life of warfarin is relatively long (about 72h), while the serum half-life of vitamin K is about 6h. Therefore, vitamin K needs to be used frequently and continuously for a long time. Heparin overdose is usually anticoagulated with protamine, but protamine only has a partial reversal effect on low molecular weight heparin and has no antidote activity on heparin derivatives (such as fondaparinux). The side effects caused by protamine are related to immunity and inflammation, and may cause allergic reactions, systemic hypotension, pulmonary hypertension, bronchoconstriction and bradycardia, etc.

[0004] Some non-vitamin K-dependent oral anticoagulants used more clinically at present are drugs that directly bind to the target (activated factor II (IIa or thrombin) or activated factor X (Xa)) and antagonize their activities. These drugs are widely applicable to patients who cannot use traditional anticoagulants or have difficulties in using traditional anticoagulants. However, among these drugs, only idarucizumab, the antidote for dabigatran, has been approved for reversing its anticoagulant effect, and there is currently no specific antidote for direct FXa inhibitors such as rivaroxaban and apixaban, which are widely used. Therefore, the research on an antidote for direct FXa inhibitors is imperative.

[0005] Andexanet alfa is a modified recombinant human factor Xa in an inactivated form and can be used as a reversal agent for direct oral FXa inhibitors. This product is for patients treated with apixaban or rivaroxaban to help reverse their anticoagulant effects in the event of life-threatening or uncontrolled bleeding. However, there is a certain risk of thrombosis after treatment with andexanet alfa, and such recombinant protein drugs are expensive and have complex preparation processes.

[0006] Joost C.M. 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 in vitro tests. Its reversal effect on rivaroxaban and apixaban was evaluated in vivo through a mouse tail amputation model and a rabbit Wessler venous stasis model. The results showed that the administration of oral anticoagulants such as rivaroxaban and apixaban would increase the bleeding time by 3 to 4 times in this model, and OKL-1111 reversed this effect with an efficiency of 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 inactivated FXa and can bind to direct FXa inhibitors with an affinity comparable to that of native FXa. The researchers determined the binding potency (Kd) of r-Antidote to three direct FXa inhibitors (betrixaban, rivaroxaban, and apixaban) and compared them with the inhibition of purified human FXa using a peptide substrate. It was found that it dose-dependently reversed the inhibitory activity of small molecule FXa inhibitors, but in the absence of inhibitors, it did not change the rate of Xa cleavage of the peptide substrate. Through in vitro clotting tests, r-Antidote reversed the anticoagulant effect of rivaroxaban in human plasma, and further studied the function of r-Antidote in restoring hemostasis in a rat tail amputation model and a rabbit liver laceration model. The results showed that r-Antidote could significantly reverse the increase in bleeding volume and prolongation of bleeding time caused by rivaroxaban, proving its effect in reversing rivaroxaban. However, the cost of this technology is relatively high, and the use of recombinant proteins may cause side effects such as allergies, making it difficult to be widely applied.

[0007] In such a context, 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] Due to the small size and extremely strong hydrophobicity of direct FXa inhibitors such as rivaroxaban and apixaban, they can form inclusion complexes with cyclodextrins, thereby dissociating them from the FXa binding site and reversing their anticoagulant effects. However, the spontaneous inclusion binding force between existing common cyclodextrins and inhibitors such as rivaroxaban is weak, and it can only play a certain solubilizing role, unable to completely include them and block their effects. In the present invention, an oligocyclodextrin is prepared by reacting crosslinking agent epichlorohydrin with β-cyclodextrin. It not only has the characteristics of monomeric cyclodextrin molecules (such as a hollow cage-like structure and the ability to interact with the interior of the molecule), but also has the characteristics of polymers (such as high molecular weight and chain-like structure). It can detach the drug from the binding site on FXa factor through the inclusion effect of the hydrophobic cavity, reverse its anticoagulant effect, so as to promote the timely recovery of normal blood coagulation function when there is an overdose of rivaroxaban.

[0009] In the first aspect of the present invention, an oligocyclodextrin is provided. The raw materials for preparing the oligocyclodextrin include cyclodextrin and a crosslinking agent; the molar ratio of the cyclodextrin to the crosslinking agent is 1:(8 - 30); examples can be 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 crosslinking agent is 1:(8 - 25); the prepared oligocyclodextrin has strong safety.

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

[0012] Preferably, the cyclodextrin is β-cyclodextrin

[0013] Preferably, the crosslinking agent is epichlorohydrin.

[0014] In the present invention, the degree of crosslinking and molecular weight of the oligocyclodextrin are regulated by exploring the feeding ratio of epichlorohydrin to β-cyclodextrin. Through a large number of experimental explorations, it is found that when the molar ratio of β-cyclodextrin to epichlorohydrin is 1:(8 - 30), the prepared oligocyclodextrin has a stronger reversing effect on rivaroxaban.

[0015] In some preferred embodiments, the preparation steps of the oligocyclodextrin include: adding β-cyclodextrin to an alkaline solution, heating to 55 - 65 °C, dropping the crosslinking agent, reacting for 1.5 - 2.5 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, then performing membrane dialysis and drying to obtain the oligocyclodextrin product.

[0016] The equation of this reaction is as follows. Under alkaline conditions, the hydroxyl groups (-OH) of cyclodextrin react with the epoxy groups of epichlorohydrin to form new chemical bonds. Further through the reaction processes of cross-linking, self-polymerization, and hydrolysis, oligocyclodextrin is generated. The prepared oligocyclodextrin not only has the characteristics of monomeric cyclodextrin molecules (such as a hollow cage-like structure and the ability to interact with the interior of the molecule), but also has polymer characteristics (such as high molecular weight and chain-like structure).

[0017]

[0018] Examples of the alkaline solution include aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous calcium hydroxide solution, ammonia water, etc.

[0019] Preferably, the alkaline solution is aqueous sodium hydroxide solution.

[0020] More preferably, the concentration of the aqueous sodium hydroxide solution is 100 - 200 g / L.

[0021] More preferably, the preparation steps of the oligocyclodextrin include: adding β-cyclodextrin to the alkaline solution, heating to 60°C, dropping the cross-linking agent, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH, then performing membrane dialysis and drying to obtain the oligocyclodextrin product.

[0022] Preferably, the molecular weight of the filtration membrane for membrane dialysis is 15 - 25 kDa.

[0023] More preferably, the molecular weight of the filtration membrane for membrane dialysis is 20 kDa.

[0024] Preferably, the pH is adjusted to neutral. More preferably, the pH is adjusted to neutral using aqueous hydrochloric acid solution.

[0025] Examples of the drying means include vacuum drying, oven drying, freeze drying, etc.; preferably freeze drying.

[0026] The second aspect of the present invention provides an application of oligocyclodextrin, and the oligocyclodextrin is applied as an antidote.

[0027] Note: An antidote refers to a substance that can interact with a specific drug (such as the anticoagulant rivaroxaban), thereby reducing or eliminating the pharmacological effect of the drug (such as anticoagulant effect), and thus reversing its biological activity or toxicity.

[0028] In some preferred embodiments, the object of reversal of the antidote is rivaroxaban.

[0029] 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 aid in the coagulation scenario. For example, when it is necessary to restore the coagulation function during a surgical procedure, cyclodextrin inclusion complex of hemostatic drugs can be injected into the patient to restore coagulation, aiming to address the problem of emergency postoperative massive bleeding and inability to coagulate. The present invention unexpectedly discovers that oligocyclodextrin can spontaneously include rivaroxaban (an anticoagulant drug, which can cause inability to coagulate and is mostly used by patients with thrombosis) through its hydrophobic cavity, and the cross-linked polymer network firmly binds rivaroxaban to oligocyclodextrin, dissociates it from the FXa site, and enables it to be rapidly cleared from the blood, thus playing the role of promoting the restoration of coagulation function.

[0030] In some preferred embodiments, the mass ratio of the oligocyclodextrin to rivaroxaban is 1:(4 - 20).

[0031] In some preferred embodiments, when the oligocyclodextrin is used as an antidote, its administration method is injection; specifically, the oligocyclodextrin and the prepared physiological saline are mixed into a mixed solution and then injected. More preferably, the concentration of oligocyclodextrin in the mixed solution is 0.5 - 2 mg / kg, and further preferably 1 mg / kg.

[0032] Beneficial effects:

[0033] The present invention provides an oligocyclodextrin and its application, having the following advantages:

[0034] (1) In the present invention, cross-linked oligocyclodextrin is generated by the reaction of epichlorohydrin and β-cyclodextrin, which can not only maintain the inherent macrocyclic structure and functional hydroxyl groups of cyclodextrin, but also have the properties of a polymer, showing a high degree of "integration" and "synergy" effects. Oligocyclodextrin combines the advantages of both cross-linked polymers and cyclodextrin, and also endows the cross-linked cyclodextrin with special physical and chemical properties.

[0035] (2) The present invention uses self-made oligocyclodextrin to reverse the anticoagulant rivaroxaban, which is a material that plays a role in promoting blood coagulation. Oligocyclodextrin spontaneously includes rivaroxaban through its hydrophobic cavity, and the cross-linked polymer network firmly binds rivaroxaban to oligocyclodextrin, dissociates it from the FXa site, and enables it to be rapidly cleared from the blood, thus playing the role of promoting the restoration of coagulation function.

[0036] (3) The present invention uses the mature auxiliary material β-cyclodextrin as a raw material. The synthesized oligocyclodextrin has no side effects such as allergies that may be caused by recombinant protein drugs, has high biocompatibility, and can still maintain high safety even when administered at a large dose; it is easy to implement.

[0037] (4) The synthesis route of the present invention is simple, the preparation process is convenient, the raw materials are inexpensive, and it has extremely high economic application value.

[0038] (5) The present invention fills the gap in the lack of corresponding antidotes for current oral anticoagulants and has great potential for market application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 . Reaction schematic diagram of oligocyclodextrin; Figure 1 In (a) is the cross-linking process, (b) is the self-polymerization process, and (c) is the degradation process;

[0040] Figure 2 . Comparison chart of in vitro blood coagulation test results; Figure 2 In A-G correspond to different oligocyclodextrins, 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 Comparative Example 2; the information of samples a-j is shown in Table 1;

[0041] Figure 3 . Chart of blood coagulation index test results; Figure 3 In a-l 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 Comparative Example 1, h corresponds to Comparative Example 2, i corresponds to Example 1, j corresponds to GAMA-CD, k corresponds to SBE-γ-CD, and l corresponds to γ-CD; among them, the triangles in the figure represent experimental data points;

[0042] Figure 4 . Chart of blood coagulation index test results of RIV; among them, "**" indicates p < 0.01, and "****" indicates p < 0.0001;

[0043] Figure 5 . Results of thromboelastogram experiment; Figure 5 In a is RWB, b is RWB + RIV, c is RWB + RIV + 100 μM CCD, and D is RWB + RIV + 200 μM CCD;

[0044] Figure 6 . Comparison chart of clotting time; among them, "*" indicates p < 0.1, and "**" indicates p < 0.01;

[0045] Figure 7 . Comparison chart of comprehensive clotting index; among them, "**" indicates p < 0.01, and "***" indicates p < 0.001;

[0046] Figure 8 . Comparison chart of fibrin function; among them, "*" indicates p < 0.1, and "**" indicates p < 0.01;

[0047] Figure 9 .1 H-NMR test results; Figure 9 In it, A1 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 the molar mass ratio;

[0048] Figure 10 .ITC measurement result graph; among them, the dots in the graph represent experimental data points;

[0049] Figure 11 .Prothrombin time measurement result graph of BALB / c mice; Figure 11 In it, a is the PT measurement result after intragastric administration of rivaroxaban. The black line in the graph is the control group, the red line is the rivaroxaban group, and the green, purple, pink, and blue lines are all the rivaroxaban + oligocyclodextrin groups, where the CCD concentrations are 0.5 g / kg, 1.0 g / kg, 1.5 g / kg, and 2.0 g / kg respectively; Figure 11 In it, b is the PT measurement result after injection of CCD; Figure 11 In it, c is the coagulation index after injection of CCD; Figure 11 The triangles in a in it represent experimental data points, Figure 11 The dots, squares, triangles, inverted triangles, and rhombuses in b in it represent experimental data points, Figure 11 The triangles in c in it represent experimental data points; "*" represents p < 0.1, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001;

[0050] Figure 12 .BALB / c mouse tail bleeding experiment result graph; among them, the dots, squares, triangles, inverted triangles, and rhombuses represent experimental data points; "****" represents p < 0.0001;

[0051] Figure 13 .BALB / c mouse liver bleeding experiment result graph; among them, "*" represents p < 0.1, "****" represents p < 0.0001;

[0052] Figure 14 .BALB / c mouse growth curve; among them, the dots, squares, triangles, inverted triangles, and rhombuses represent experimental data points;

[0053] Figure 15 .BALB / c mouse blood routine test results; among them, the dots, squares, triangles, inverted triangles, and rhombuses represent experimental data points;

[0054] Figure 16 . Biochemical results of BALB / c mice; among them, dots, squares, triangles, inverted triangles, and diamonds represent experimental data points;

[0055] Figure 17 . Organ index results of BALB / c mice; among them, dots, squares, triangles, inverted triangles, and diamonds represent experimental data points;

[0056] Figure 18 . Observation results of organs under optical microscope; 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 in turn; the scale in the figure is 100μm;

[0057] Figure 19 . Hemolysis test results; Figure 19 A - F in correspond to different oligocyclodextrins, 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. Specific implementation manners

[0058] The reaction schematic diagram for preparing oligocyclodextrin in the embodiments of the present invention is shown in Figure 1 .

[0059] Note: Unless otherwise specified, the solvent of the solutions involved in the present invention is water; and, the raw materials used are all commercially available.

[0060] Examples

[0061] Example 1

[0062] This example provides an oligocyclodextrin, and the preparation raw materials of the oligocyclodextrin include β - cyclodextrin (β - CD) and a cross - linker (epichlorohydrin); the molar ratio of the β - cyclodextrin to the cross - linker is 1:4.

[0063] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β - cyclodextrin, heating to 60°C, dropping 2.75 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with a 5 wt% HCl aqueous solution, then dialyzing with a 20 kDa membrane, and freeze - drying to obtain an oligocyclodextrin product.

[0064] Example 2

[0065] This example provides an oligocyclodextrin, and the preparation raw materials of the oligocyclodextrin include β - cyclodextrin and a cross - linker (epichlorohydrin); the molar ratio of the β - cyclodextrin to the cross - linker is 1:8.

[0066] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 5.5 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane and freeze-drying to obtain the oligomeric cyclodextrin product.

[0067] Example 3

[0068] This example provides an oligomeric cyclodextrin. The raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:10.

[0069] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 6.88 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane and freeze-drying to obtain the oligomeric cyclodextrin product.

[0070] Example 4

[0071] This example provides an oligomeric cyclodextrin. The raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:15.

[0072] The preparation steps of the oligomeric cyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 10.31 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane and freeze-drying to obtain the oligomeric cyclodextrin product.

[0073] Example 5

[0074] This example provides an oligomeric cyclodextrin. The raw materials for preparing the oligomeric cyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:20.

[0075] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 13.75 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane, and freeze-drying to obtain the oligocyclodextrin product.

[0076] Example 6

[0077] This example provides an oligocyclodextrin. The raw materials for preparing the oligocyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:25.

[0078] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 17.19 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane, and freeze-drying to obtain the oligocyclodextrin product.

[0079] Example 7

[0080] This example provides an oligocyclodextrin. The raw materials for preparing the oligocyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:30.

[0081] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 20.63 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane, and freeze-drying to obtain the oligocyclodextrin product.

[0082] Comparative Example 1

[0083] This comparative example provides an oligocyclodextrin. The raw materials for preparing the oligocyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of β-cyclodextrin to the cross-linking agent is 1:0.1.

[0084] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 0.07 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane and freeze-drying to obtain the oligocyclodextrin product.

[0085] Comparative Example 2

[0086] This comparative example provides an oligocyclodextrin. The preparation raw materials of the oligocyclodextrin include β-cyclodextrin and a cross-linking agent (epichlorohydrin); the molar ratio of the β-cyclodextrin to the cross-linking agent is 1:1.

[0087] The preparation steps of the oligocyclodextrin include: dissolving 3.52 g of sodium hydroxide in 25 ml of water, then adding 10 g of β-cyclodextrin, heating to 60 °C, dropping 0.69 mL of epichlorohydrin, reacting for 2 h to obtain a reaction mixture; cooling the reaction mixture to room temperature, adjusting the pH to neutral with 5% HCl aqueous solution, then dialyzing with a 20 kDa membrane and freeze-drying to obtain the oligocyclodextrin product.

[0088] Performance test

[0089] The abbreviations and interpretations of some samples involved in the following tests are as follows:

[0090] CCD: Oligocyclodextrin; unless otherwise specified in Tests 1 to 8, the oligocyclodextrin samples used are from Example 2.

[0091] CD: Cyclodextrin;

[0092] β-CD: Commercially available cyclodextrin, manufacturer Macklin, CAS number: 7585-39-9;

[0093] Cationic-CD: Cationic cyclodextrin, commercially available cyclodextrin, manufacturer Zhiyuan Biology, batch number: CC20240424P;

[0094] HP-γ-CD: Commercially available cyclodextrin, manufacturer Zhiyuan Biology, batch number: HG20230709;

[0095] SBE-γ-CD: Commercially available cyclodextrin, manufacturer Zhiyuan Biology, product batch number: SB20240330;

[0096] RIV: Rivaroxaban;

[0097] ECH: Epichlorohydrin.

[0098] RWB: Rat whole blood.

[0099] And, unless otherwise specified, the control samples contral involved in each test are all basal control samples.

[0100] 1. In vitro coagulation test

[0101] Test method:

[0102] 1) Anesthetize SD rats with isoflurane, collect fresh blood samples and add them to a 10 mL centrifuge tube containing an anticoagulant (3.8 wt% sodium citrate aqueous solution). The ratio of fresh blood samples to the anticoagulant is 9:1 to obtain anticoagulated blood. Keep the anticoagulated blood on ice and conduct the experiment within 4 h.

[0103] 2) Take 5 ml EP tubes, add 98 μL of anticoagulated blood to each tube, and set up ① control group, ② rivaroxaban group, and ③ rivaroxaban + oligocyclodextrin group respectively.

[0104] 3) Add 1 μL of DMSO to the control group and mix well. Add 1 μL of 2 mg / mL rivaroxaban solution (solvent DMSO) to the rivaroxaban group and the rivaroxaban + oligocyclodextrin group. The final concentration of rivaroxaban in the blood is 20 μg / mL, and mix well.

[0105] 4) Add 1 μL of normal saline to the control group and the rivaroxaban group respectively, and add 1 μL of oligocyclodextrin solution (solvent is normal saline) to the rivaroxaban + oligocyclodextrin group. The final concentrations of oligocyclodextrin in the blood are 1.25, 2.5, 5, 10, 20, 40, 80, 160 μg / mL. Mix well. Incubate in a 37 °C constant temperature incubator for 10 min.

[0106] 5) After incubation, add 20 μL of 0.2 M calcium chloride solution (solvent is water) to activate the blood to initiate coagulation, mix well again, and observe the coagulation effect after incubating in a 37 °C constant temperature incubator for 10 min. See Figure 2 . Figure 2 For the information of samples a - i in Figure 3 refer to Table 1. Then add 3 mL of deionized water along the side wall, and incubate for another 5 min to completely rupture the blood cells of the uncured blood clot and release hemoglobin, and then measure the absorbance at 540 nm. Repeat each group 3 times. Figure 3 A - G in

[0107] correspond to different oligocyclodextrins, Figure 3 .Figure 3 Ctrl in it corresponds to sample a (control group), RIV corresponds to sample b (rivaroxaban group), and 1.25 - 160 represents the concentration of oligocyclodextrin in the rivaroxaban + oligocyclodextrin group (unit: μg / mL). Figure 3 In each group of tests in, samples a - j 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 Comparative Example 1, h corresponds to Comparative Example 2, i corresponds to Example 1 (RIV 4 μg / mL), j corresponds to GAMA - CD, k corresponds to SBE - γ - CD, and l corresponds to γ - CD.

[0108] At the same time, the coagulation index of RIV was measured, and the results are shown in Figure 4 .

[0109] Table 1

[0110]

[0111] Note: In Table 1, - represents not added, and + represents added.

[0112] From Figure 2 it can be seen that adding oligocyclodextrin solutions with a series of concentrations of 1.25 - 80 μg / mL can reverse the effect of rivaroxaban in ex vivo whole blood and restore blood coagulation function. At the same time Figure 3 、 Figure 4 The results show that compared with the rivaroxaban group, the hemoglobin content in the rivaroxaban + oligocyclodextrin group decreased, the coagulation index decreased, and the blood coagulation function was restored.

[0113] 2. Rotational thromboelastometry (ROTEM) experiment

[0114] SD rats were anesthetized with isoflurane, and ex vivo blood of the rats 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 it was incubated at 37 °C for 5 minutes. The reversing agent oligocyclodextrin dissolved in physiological saline was added to make its concentration in the blood 100 μM or 200 μM, and it 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 thromboelastograph (Lepu LEPU, LEPU - Auto8) for measurement, and the measurement results are shown in Figures 5 - 8 ; at the same time, a control sample contral was set. Figure 5In this, a 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 blood coagulation, and the ordinate is the amplitude. Figure 6 In this, the coagulation time (R) refers to the time from the initiation of the coagulation system to the start of the formation of the fibrin clot (the amplitude of the TEG tracing reaches 2 mm); Figure 7 In this, the coagulation index (CI) is an index that comprehensively reflects the coagulation function of the sample, and is a comprehensive result based on three indicators: prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen level. These indicators together reflect the activity of coagulation factors in the blood and the process of fibrin formation; the fibrin function (angle) refers to the angle between the horizontal line and the tangent line drawn from the point of fibrin clot formation to the maximum curvature of the curve.

[0115] 3. 1 1H-NMR test

[0116] 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. After mixing, a magnetic stir bar was added and stirred at room temperature on a magnetic stirrer for 1 h at a stirring speed of 500 rpm. A 400M nuclear magnetic resonance spectrometer (BRUKER®, AvanceⅢ) was used for 1 1H-NMR determination. After obtaining the data, it was processed with Mestrenova®. Using the D6-DMSO solvent peak (2.5000 ppm) as a reference, the chemical shifts of the rivaroxaban characteristic peaks in the rivaroxaban + oligocyclodextrin mixture were compared with those of the rivaroxaban solution characteristic peaks. Through 1 the changes and displacements of the rivaroxaban characteristic peaks in 1H-NMR to determine whether rivaroxaban and oligocyclodextrin spontaneously underwent an inclusion complexation. The test results are shown in Table 2 and Figure 9 A1~A3 in this. The results confirmed that rivaroxaban and oligocyclodextrin spontaneously underwent an inclusion complexation. At the same time, the mass spectrometry results of mixtures of rivaroxaban and β-CD mixed at different concentrations were measured, and the results are shown in Figure 9 B1~B6 in this.

[0117] Table 2

[0118]

[0119] 4. Isothermal titration calorimetry (ITC) experiment

[0120] The in vitro affinity between oligocyclodextrin and rivaroxaban can be determined by an isothermal titration calorimeter (MicroCal®, PEAQ-ITC). A 2 mM oligocyclodextrin solution (solvent: PBS buffer containing 5% DMSO) was added to a 100 μM rivaroxaban sample cell (solvent: PBS buffer containing 5% DMSO) through a syringe, with 27 injections. After titration, it was analyzed using the MicroCal® PEAQ-ITC Analysis Software. The results are shown in Figure 10 . The in vitro binding force (Ks) between oligocyclodextrin and rivaroxaban was determined to be 5000 M -1 , and the enthalpy change (ΔH) was -2.18 kJ / mol, indicating that oligocyclodextrin and rivaroxaban have good in vitro affinity.

[0121] 5. Prothrombin time (PT) experiment in BALB / c mice

[0122] 5.1 PT determination of rivaroxaban + oligocyclodextrin

[0123] 1) Experimental animals: 218 male BALB / c mice.

[0124] 2) Preparation of reagents:

[0125] 20 mg / kg rivaroxaban solution (solvent: PEG400, absolute ethanol, and water mixed in a volume ratio of 3:1:1, and the dosing volume for each mouse is 0.05 mL / 10 g);

[0126] 0.5 g / kg, 1.0 g / kg, 1.5 g / kg, and 2 g / kg oligocyclodextrin solutions (solvent: normal saline, and the dosing volume for each mouse is 0.05 mL / 10 g).

[0127] 3) Experimental grouping:

[0128] Blank group: 8 mice

[0129] Rivaroxaban model group: 50 mice, 10 mice each at 1 h, 1 h 10 min, 2.5 h, 3.5 h, and 5 h

[0130] Rivaroxaban + 0.5 g / kg oligocyclodextrin group: 40 mice, 10 mice each at 1 h 10 min, 2.5 h, 3.5 h, and 5 h

[0131] Rivaroxaban + 1.0 g / kg oligocyclodextrin group: 40 mice, 10 mice each at 1 h 10 min, 2.5 h, 3.5 h, and 5 h

[0132] Rivaroxaban + 1.5 g / kg oligocyclodextrin group: 40 mice, 10 mice each at 1 h 10 min, 2.5 h, 3.5 h, and 5 h

[0133] Rivaroxaban + 2.0 g / kg oligocyclodextrin group: 40 mice, 10 mice each at 1 h 10 min, 2.5 h, 3.5 h, and 5 h

[0134] Note: The time recorded for each group starts from the administration of rivaroxaban by gavage. That is, 1 h represents one hour after the administration of rivaroxaban by gavage. Oligocyclodextrin was injected 1 h after the administration of rivaroxaban, and blood was collected 1.5 h, 2.5 h, and 4 h after the injection of oligocyclodextrin. Therefore, it corresponds to the 2.5 h, 3.5 h, and 5 h groups respectively.

[0135] 4) Experimental procedure:

[0136] First step: Before gavaging rivaroxaban, blood was collected from the mouse eyeballs to obtain the PT (s) of the mice in the normal state, and 10 mice were treated.

[0137] Second step: Both the rivaroxaban group and the rivaroxaban + oligocyclodextrin group were gavaged with a 20 mg / kg rivaroxaban solution by oral administration.

[0138] Third step: One hour after gavaging in the rivaroxaban model group, blood was collected from the mouse eyeballs. A total of 10 mice were used to measure PT. At the same time, in the rivaroxaban + oligocyclodextrin group, mice were injected with oligocyclodextrin solution via the tail vein according to the oligocyclodextrin concentrations of 0.5 g / kg group (1 h 10 min, 2.5 h, 3.5 h, and 5 h groups), 1.0 g / kg group (1 h 10 min, 2.5 h, 3.5 h, and 5 h groups), 1.5 g / kg group (1 h 10 min, 2.5 h, 3.5 h, and 5 h groups), 2.0 g / kg group (1 h 10 min, 2.5 h, 3.5 h, and 5 h groups), with a total of 160 mice (40 mice in each group).

[0139] Fourth step: At each time point, mice in each group were anesthetized with isoflurane and then the whiskers of the mice were cut off for eyeball enucleation and blood collection. Blood was collected from the mouse eyeballs. Immediately after blood collection, the supernatant was obtained by centrifugation at 3000 rpm and 4 °C, and PT was measured.

[0140] 5) Specific method for PT measurement:

[0141] Collect mouse eyeball blood using an anticoagulant tube, gently invert the EP tube up and down, and then immediately place it on ice. The storage time under ice bath is within 4 h. Centrifuge at 3000 r / min for 15 min within 4 h, and then use a pipette to aspirate the supernatant plasma. Use a prothrombin (PT) detection kit (Yuanye Bio®, R24682 - 100T), and according to the method of the PT kit, detect and record the PT value of the sample. The results are shown in Figure 11 a in

[0142] AsFigure 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.

[0143] 5.2 Oligomeric cyclodextrin PT determination

[0144] 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 Figure 11 PT was measured at 2.5h on the horizontal axis of a in the figure. The results are shown in Figure 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 Figure 11 In center c, the results show that oligomeric cyclodextrin (CCD) alone cannot restore normal coagulation.

[0145] 6. BALB / c mouse tail bleeding liver bleeding model experiment

[0146] 1) Experimental animals: 220 male BALB / c mice.

[0147] 2) Experimental reagents:

[0148] 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);

[0149] 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).

[0150] 3) Experimental groups:

[0151] Blank group: 8

[0152] Rivaroxaban model group

[0153] Rivaroxaban + 0.5g / kg oligomeric cyclodextrin group

[0154] Rivaroxaban + 1.0g / kg oligomeric cyclodextrin group

[0155] Rivaroxaban + 1.5g / kg oligomeric cyclodextrin group

[0156] Rivaroxaban + 2.0g / kg oligomeric cyclodextrin group

[0157] 4) Experimental procedure:

[0158] Step 1: The mice in the blank group were intragastrically administered normal saline (administration volume: 0.05 mL / 10 g), and the mice in the rivaroxaban model group and the rivaroxaban + oligocyclodextrin group were intragastrically administered rivaroxaban solution (administration volume: 0.05 ml / 10 g) respectively.

[0159] Step 2: The average tail diameter of the mice at 2 cm from the tail tip was measured using a vernier caliper to obtain the average diameter D.

[0160] Step 3: The mice were anesthetized with isoflurane. 60 minutes after intragastric administration of rivaroxaban, the tail of the mice at the position with diameter D was cut off using a scalpel. 1 mL of water was added to a 2 mL EP tube, and the EP tube was placed at the tail of the tail-cut mice. The wound was extended below the liquid surface to collect blood for 30 minutes. After dilution by 10 times, the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to determine the amount of tail bleeding (the higher the absorbance, the higher the surface bleeding volume). The test results are shown in Figure 12 。

[0161] Step 4: Liver bleeding: 10 minutes after collecting tail blood, the abdomen was immediately spread open with surgical scissors. A 1 cm long and 3 mm deep wound was made on the left liver lobe of the rats. After pre-weighing the filter paper and cotton ball, the filter paper was placed under the liver lobe, and the blood was collected with a clean cotton ball once every 4 s. The weight of the filter paper and cotton ball was weighed to determine the blood loss in 15 minutes. The test results are shown in Figure 13 。

[0162] Figure 12 、 13 As shown in, after oral intragastric administration of rivaroxaban, the liver bleeding volume and tail bleeding volume of the mice increased significantly. At the time point when the effect of rivaroxaban was obvious, the corresponding dose of oligocyclodextrin was injected, and both the liver bleeding volume and tail bleeding volume of the mice decreased, and could be restored to near the normal level, indicating that the oligocyclodextrin of the present invention has a significant reversal effect.

[0163] 7. Safety evaluation of oligocyclodextrin

[0164] Male BALB / C mice weighing 24 - 28 g were grouped. In the control group, normal saline was injected intravenously, and in the administration group, mice were injected intravenously with an oligocyclodextrin solution (the solvent was normal saline). Their body weights were measured daily, and their general behaviors were observed. Continuous monitoring was carried out for 21 days, and after 21 days, the mice were euthanized. Blood samples were collected for hematological analyses such as whole blood cells, red blood cells, platelets, and hemoglobin. The concentrations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA) were measured using a kit. The main organs of the mice were isolated, weighed, and the organ index was calculated (the organ index is the ratio of the weight of each organ of a mouse to its body weight). The test results are shown in Figure 14 、 Figure 15 、 Figure 16 、 Figure 17 。Meanwhile, the heart, liver, spleen, lung, and kidney tissue sections of the mice were stained with H&E (hematoxylin - eosin staining), and the imaging results were obtained using an integrated cell imaging analysis system (EVOS M7000) as shown in Figure 18 , Figure 18 where each column corresponds to the same organ, and the five columns from left to right are the heart, liver, spleen, lung, and kidney in sequence; the scale bar in the figure is 100 μm. The experimental results show that the oligocyclodextrin prepared by the present invention has good safety at an addition amount of 0.5 - 2.0 g / kg.

[0165] 8. Hemolysis experiment

[0166] 1) Blood was taken from an SD rat and collected in an anticoagulation tube containing an anticoagulant (3.8% sodium citrate aqueous solution). The ratio of fresh blood sample to anticoagulant was 9:1. The supernatant plasma and platelets were removed by centrifugation at 3000 rpm and 4 °C for 15 minutes to obtain red blood cells.

[0167] 2) The red blood cells were resuspended in normal saline 3 - 4 times under the same conditions as centrifugation.

[0168] 3) A 2% red blood cell suspension (2 mL red blood cells + 98 mL normal saline) was prepared with normal saline.

[0169] 4) 500 μL of the red blood cell suspension was added to an EP tube, 500 μL of red blood cell lysate was added to the positive control, 500 μL of normal saline was added to the negative control group, and a series of concentration gradients of oligocyclodextrin (500 μL) were added to the experimental group. After mixing evenly, the samples were incubated at 37 °C for 3 h.

[0170] 5) The samples were centrifuged at 3000 rpm and 4 °C for 15 minutes. The supernatant was taken and measured using an enzyme - linked immunosorbent assay (ELISA) reader at a wavelength of 540 nm.

[0171] 6) Data processing: Hemolysis rate = (sample - negative control) / (positive control - negative control) * 100%; The test results are shown inFigure 19 。 Figure 19 A - F in it correspond to different oligocyclodextrins. 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.

[0172] Note: A hemolysis rate < 5% is considered safe.

[0173] Combined with the analysis of experimental results, it can be seen that the oligocyclodextrin prepared by the present invention can effectively restore the obstruction of whole blood coagulation caused by rivaroxaban in vitro, and normalize the blood coagulation function. At the same time, it can effectively reverse the phenomena such as the prolongation of thrombin time, the decline of fibrin function, and the decline of comprehensive coagulation index caused by rivaroxaban. After adding oligocyclodextrin, the function of blood coagulation can be restored. And when oligocyclodextrin is added to mice, it can restore the prolongation of prothrombin time (PT) of mouse blood caused by rivaroxaban. The evaluation results of the mouse tail - cutting model and the mouse liver hemorrhage model show that the administration of oral anticoagulants such as rivaroxaban induces an increase in bleeding time in this model, while the intravenous injection of oligocyclodextrin can reverse this effect. The safety evaluation results show that oligocyclodextrin does not affect the growth of animal body weight in vivo. Tissue staining section evaluation and blood routine and blood biochemical tests show that it has no toxicity to the heart, liver, spleen, lung, and kidney. The oligocyclodextrin prepared by the present invention has excellent reversal effect and strong safety.

Claims

1. An application of 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; The oligomeric cyclodextrin is applied to an antidote, and the reversal object of the antidote is rivaroxaban.

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

Images