Uric acid adsorbent as well as preparation method and application thereof

Abstract

The invention provides a uric acid adsorbent as well as a preparation method and application thereof, and the preparation method of the uric acid adsorbent comprises the following steps: grafting PEGylated uricase on carboxyl-containing porous resin to prepare pretreated porous resin; uniformly mixing the second PEGylated uricase solution and the collodion solution to prepare a coating solution; and heating and stirring the pretreated porous resin and the coating solution at a preset temperature, so that the PEGylated uricase is immobilized on the pretreated porous resin, and the uric acid adsorbent is prepared. The uric acid adsorbent provided by the invention has good adsorption performance on uric acid in blood, can reduce side effects and complications of an existing uric acid removal method, and has good safety.

Description

Technical Field

[0001] The present invention relates to the technical field of blood purification, and in particular to a uric acid adsorbent and a preparation method and application thereof. Background Art

[0002] Gout is a disease caused by a disorder of uric acid metabolism. Its main characteristic is elevated blood uric acid levels, which lead to the deposition of urate crystals in joints and soft tissues, triggering acute inflammation and recurrent pain. Currently, the main treatments for uric acid in the blood are medication and surgical removal.

[0003] Existing drug treatments mainly reduce blood uric acid levels by inhibiting uric acid production (such as the use of xanthine oxidase inhibitors) or promoting uric acid excretion (such as the use of URAT1 inhibitors). However, these drugs are often accompanied by significant side effects, such as kidney damage, liver toxicity, and bone marrow suppression, and have limited efficacy, and cannot fundamentally solve the problem of urate crystal deposition in gout patients. In addition, some drugs, such as Febuxostat, may also cause complications such as hypokalemia.

[0004] Although blood purification can currently be used to absorb uric acid in the blood to reduce the uric acid content in the blood, these adsorption methods have the problem of non-specific adsorption. While adsorbing the harmful substance uric acid, some beneficial body fluid components with a similar molecular weight to uric acid are also adsorbed, which leads to the loss of beneficial body fluid components in the conductor blood and causes some potential health risks. Summary of the Invention

[0005] The present invention aims to provide a uric acid adsorbent and a preparation method and application thereof, so as to solve the problems of low safety and poor effect of existing methods in reducing uric acid in blood.

[0006] To solve the above problems, the first aspect of the present invention provides a method for preparing a uric acid adsorbent, comprising:

[0007] Grafting PEGylated uricase onto a porous resin containing carboxyl groups to prepare a pretreated porous resin;

[0008] The second PEGylated uricase solution and the collodion solution are mixed evenly to prepare a coating solution;

[0009] The pretreated porous resin and the coating solution are heated and stirred at a preset temperature to immobilize the PEGylated uricase on the pretreated porous resin, thereby preparing a uric acid adsorbent.

[0010] The second aspect of the present invention provides a uric acid adsorbent prepared by the preparation method described in the first aspect.

[0011] The third aspect of the present invention provides an application of a uric acid adsorbent, wherein the uric acid adsorbent prepared by the preparation method described in the first aspect, or the uric acid adsorbent described in the second aspect, is used as an adsorption filler in a hemoperfusion device, a hemoadsorber, or a plasma adsorber.

[0012] The uric acid adsorbent, preparation method, and application thereof disclosed herein utilize a porous resin containing carboxyl groups as a carrier and carboxyl groups as a connecting structure. PEGylated uricase is chemically grafted onto the surface of the porous resin via an esterification reaction, thereby immobilizing the PEGylated uricase on the surface of the pretreated porous resin. Subsequently, a coating solution is prepared using a PEGylated uricase solution and a collodion solution. The coating solution and the pretreated porous resin are heated and stirred to immobilize the PEGylated uricase on the pretreated porous resin. The PEGylated uricase can be immobilized on the surface and within the pores of the pretreated porous resin by physical encapsulation. Physical encapsulation of the PEGylated uricase can prevent the shedding of particles and avoid the occurrence of thromboembolism. Furthermore, physical encapsulation with collodion, where the collodion is coated on the surface and within the pores of the porous resin, can improve the biocompatibility and safety of the porous resin and significantly enhance the overall performance of the uric acid adsorbent. The present invention combines chemical grafting with physical encapsulation to not only improve the firmness of the bond between the PEGylated uricase and the porous resin, but also immobilize the PEGylated uricase on the surface and within the pores of the porous resin. This fully utilizes the structure of the porous resin and greatly increases the immobilized capacity of the PEGylated uricase on the porous resin, enabling the uric acid adsorbent to accurately adsorb uric acid from the blood. Furthermore, the PEGylated uricase chemically grafted to the surface of the porous resin allows direct contact with the uric acid in the blood, degrading it. The physically encapsulated PEGylated uricase provides a "sustained release" effect, extending the duration of action of the PEGylated uricase while adsorbing uric acid, thereby further degrading uric acid in the blood. The uric acid adsorbent provided by the present invention has excellent adsorption performance for uric acid in the blood, and the PEGylated uricase has advantages such as a long half-life and a low immune response, which can reduce the side effects and complications of existing uric acid removal methods. The uric acid adsorbent has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a process flow chart for preparing a uric acid adsorbent provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0014] Although the prior art can use electrostatic spraying to spray bioglue (medical-grade n-octyl α-cyanoacrylate) onto polystyrene composite microspheres or activated carbon particles, and then wrap the uricase concentrate thereon to obtain immobilized uricase particles to achieve the purpose of reducing uric acid, uricase is almost insoluble in water. Directly using a uricase aqueous solution for spraying on the resin surface results in a low effective concentration of uricase, and the degradation effect on uric acid is not obvious. More importantly, uricase is a protein derived from microorganisms (such as Bacillus subtilis) or mammals (such as pig-baboon chimeras), and has a low homology with the human body's natural uricase, which makes it easy for the human immune system to recognize it as a foreign antigen, thereby triggering immune response problems. It is reported that the use of such immobilized uricase particles causes about 40% of patients to develop anti-drug antibodies during treatment. Therefore, directly using uricase to degrade uric acid is prone to immune problems. Furthermore, the stability of uricase is affected by various factors, which can affect its activity and thus its degradation of uric acid. Furthermore, uricase has a half-life of only about 45 minutes in the body, which makes it difficult to achieve maximum adsorption in such a short time, resulting in poor uric acid adsorption performance of the immobilized uricase particles. Furthermore, although bioglue (medical-grade n-octyl α-cyanoacrylate) has low toxicity, its strong hemostatic effect can also cause adverse reactions upon contact with blood, making it unsuitable for use in blood purification technology.

[0015] In order to solve the above-mentioned problems of existing uric acid adsorbents, the present invention provides a uric acid adsorbent and its preparation method and application. The uric acid adsorbent has a good adsorption effect on uric acid in the blood and is safer than the existing technology.

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0017] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0018] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, and other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0019] In addition, although the present invention describes the various steps in the preparation in the form of S110, S120 and S130, this description is only for ease of understanding. The form of S110, S120 and S130 does not limit the order of the steps.

[0020] Figure 1 The process flow chart for preparing uric acid adsorbent provided in the examples of this application. Figure 1 As shown, the first aspect of the embodiment of the present application provides a method for preparing a uric acid adsorbent, comprising:

[0021] Step S110: Grafting PEGylated uricase onto a porous resin containing carboxyl groups to prepare a pretreated porous resin.

[0022] Specifically, PEGylated uricase is prepared into a first PEGylated uricase solution, a porous resin containing a carboxyl group and dicyclohexylcarbodiimide are added to the first PEGylated uricase solution, and then a 4-dimethylaminopyridine solution is dropped into it. The reaction is carried out in an ice bath to graft the PEGylated uricase onto the porous resin to prepare a pretreated porous resin.

[0023] In this embodiment, PEGylated uricase is first prepared into a first PEGylated uricase solution, and then a porous resin containing carboxyl groups and dicyclohexylcarbodiimide are added to the first PEGylated uricase solution. Dicyclohexylcarbodiimide, as a dehydrating agent, can first activate the carboxyl groups in the porous resin. Then, under the action of 4-dimethylaminopyridine, the activated carboxyl groups can undergo an esterification reaction with the hydroxyl groups in the PEGylated uricase, thereby chemically bonding the PEGylated uricase to the surface of the porous resin. This improves the tightness of the bond between the PEGylated uricase and the porous resin, and also immobilizes a certain amount of PEGylated uricase on the pretreated porous resin surface.

[0024] PEGylated uricase is a urate oxidase modified with polyethylene glycol (PEG). After PEGylation, uricase is essentially non-immunogenic, significantly reducing the immunogenicity of uricase and retaining approximately 75% of the uric acid decomposition activity of unmodified uricase. PEGylated uricase is also more soluble and can be mixed with a variety of solvents.

[0025] In this embodiment, the porous resin containing carboxyl groups refers to a porous resin containing a certain amount of carboxyl groups, and the porous resin itself has a large specific surface area and a rich pore structure, which is conducive to adsorption. In this embodiment, the content of carboxyl groups in the porous resin is not further limited, and those skilled in the art can select it according to actual conditions.

[0026] Based on the above embodiment, as an optional embodiment, the porous resin containing carboxyl groups is a carbonized resin containing carboxyl groups, and the carbonized resin containing carboxyl groups is a porous spherical carbonized resin with a particle size range of 0.3 mm to 1.0 mm and a specific surface area greater than 300 m 2 / g, for example: the specific surface area is 320m 2 / g、340m 2 / g、360m 2 / g、380m 2 / g, 400m 2 / g, 450m2 / g or 500m 2 / g, etc., and its average pore size (BET) is 2nm to 6nm. Thus, the above-mentioned carbonized resin containing carboxyl groups has both carboxyl groups and good adsorption capacity, which is conducive to adsorbing substances such as cytokines in blood, and its high safety can be used in the field of blood purification technology. Of course, those skilled in the art can also select other types of porous resins according to actual conditions, as long as it can be ensured that the porous resin contains carboxyl groups, and the porous resin has a larger specific surface area and abundant pore structure, which is suitable for use in the field of blood purification technology.

[0027] Based on the above embodiment, as an optional implementation manner, before adding the porous resin containing carboxyl groups and dicyclohexylcarbodiimide to the first PEGylated uricase solution, the method further includes:

[0028] The porous resin containing carboxyl groups is dried to reduce the water content of the porous resin containing carboxyl groups to no more than (i.e., less than or equal to) 20 wt %. Thus, drying the porous resin containing carboxyl groups before use can prevent residual moisture from interfering with the esterification reaction between the carboxyl groups and the hydroxyl groups in the PEGylated uricase, thereby facilitating the esterification reaction between the carboxyl groups and the hydroxyl groups in the PEGylated uricase.

[0029] Based on the above embodiment, as an alternative embodiment, PEGylated uricase is dissolved in an anhydrous DMF (dimethylformamide) solution and stirred until the PEGylated uricase is completely dissolved to prepare a first PEGylated uricase solution. Thus, using an anhydrous DMF solution to prepare the first PEGylated uricase solution facilitates the miscibility of the carboxyl-containing porous resin, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine solution in the first PEGylated uricase solution, thereby facilitating the esterification reaction between the activated carboxyl groups and the hydroxyl groups in the PEGylated uricase. As an alternative embodiment, the mass concentration of the first PEGylated uricase solution is 1% to 3%, preferably, the mass concentration of the first PEGylated uricase solution is 2%.

[0030] Based on the above embodiment, as an optional embodiment, the amount of dicyclohexylcarbodiimide used is 4‰ to 8‰ of the porous resin containing carboxyl groups. Preferably, the amount of dicyclohexylcarbodiimide used is 6‰ of the porous resin containing carboxyl groups. This is conducive to the dicyclohexylcarbodiimide fully activating the carboxyl groups on the porous resin.

[0031] In this embodiment, the mass ratio of the first PEGylated uricase solution to the porous resin containing carboxyl groups is not further limited. Those skilled in the art can determine the mass ratio based on the content of carboxyl groups on the porous resin, as long as the amount of PEGylated uricase used is greater than the amount of carboxyl groups on the porous resin.

[0032] In this embodiment, the ice bath temperature is 0° C. to 5° C. Carrying out the reaction at this temperature is conducive to the esterification reaction between the activated carboxyl group and the hydroxyl group in the PEGylated uricase, and improves the efficiency of the esterification reaction.

[0033] Step S120: Evenly mix the second PEGylated uricase solution and the collodion solution to prepare a coating solution.

[0034] Specifically, a second PEGylated uricase solution and a collodion solution are prepared respectively; the second PEGylated uricase solution and the collodion solution are mixed in a volume ratio of 1:1 to 1:3, and stirred until the mixture becomes clear to prepare a coating solution, wherein the volume content of the PEGylated uricase in the coating solution is 2.5‰ to 2%.

[0035] In this embodiment, by first separately preparing a second PEGylated uricase solution and a collodion solution, and then mixing the second PEGylated uricase solution and the collodion solution to prepare a coating solution, a homogeneous phase of PEGylated uricase and collodion is formed, allowing the PEGylated uricase to be evenly distributed in the collodion. The collodion, having a certain viscosity, can encapsulate the PEGylated uricase, thereby immobilizing the PEGylated uricase. In this embodiment, the volume ratio of the second PEGylated uricase solution to the collodion solution is within the aforementioned range, ensuring that the PEGylated uricase is encapsulated by the collodion, improving the tightness of the PEGylated uricase attached to the pretreated porous resin, and ensuring that the coating solution subsequently forms a structurally stable film layer on the surface and within the pores of the porous resin. In this embodiment, the volume content of PEGylated uricase in the coating solution is 2.5‰ to 2%, which is beneficial for increasing the immobilized capacity of PEGylated uricase on the uric acid adsorbent, thereby improving the adsorption performance of uric acid.

[0036] Based on the above embodiment, as an optional embodiment, a second PEGylated uricase solution is prepared, comprising:

[0037] The PEGylated uricase is dissolved in a buffer solution to prepare an intermediate solution; a mixture of anhydrous ethanol and water is added to the intermediate solution and stirred to mix uniformly to prepare a second PEGylated uricase solution. Thus, by first dissolving the PEGylated uricase in the buffer solution, a stable pH environment is provided for the PEGylated uricase, thereby improving the stability of the PEGylated uricase. The intermediate solution is then stirred to mix uniformly with the mixture of anhydrous ethanol and water. Alternating the two solvents, the buffer solution and the anhydrous ethanol and water mixture, facilitates the full dissolution of the PEGylated uricase, subsequently facilitating the mutual dissolution of the PEGylated uricase and the collodion, and ensuring that the concentration of the PEGylated uricase in the second PEGylated uricase solution is within an appropriate range, thereby improving the stability of the PEGylated uricase. In this embodiment, the mass concentration of the intermediate solution is no greater than 5 mg / mL, and the mass concentration of the second PEGylated uricase solution is no less than 1 mg / mL. That is, the mass concentration of the PEGylated uricase in the second PEGylated uricase solution is W, and 1 mg / mL≤W≤5 mg / mL.

[0038] Based on the above example, as an optional embodiment, the buffer solution is trimethylammonium hydrochloride solution (i.e., Tris-HCl buffer solution), the concentration of the Tris-HCl buffer solution is 0.1 mol / L to 0.2 mol / L, and the pH value of the Tris-HCl buffer solution is 7.2 to 7.4. Therefore, the choice of Tris-HCl buffer solution as the buffer solution not only helps maintain the stability of the PEGylated uricase, but also has minimal interference with the PEGylated uricase, thus not affecting its normal function.

[0039] Based on the above embodiment, as an alternative embodiment, the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water does not exceed 50%. For example, the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water is 40%, 45%, and 50%. Preferably, the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water is 50%. Increasing the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water helps shorten the dissolution time of PEGylated uricase. Further increasing the volume fraction of anhydrous ethanol may cause precipitation of PEGylated uricase. Therefore, limiting the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water to the above range helps shorten the dissolution time of PEGylated uricase while preventing precipitation of PEGylated uricase.

[0040] Based on the above embodiment, as an optional implementation manner, preparing the collodion solution includes:

[0041] Collodion is added to a mixture of anhydrous ethanol and ether and stirred to obtain a collodion solution. The collodion solution has a mass concentration of 3% to 5%. By mixing the collodion with the mixture of anhydrous ethanol and ether, the collodion can be diluted to a suitable concentration, thereby avoiding excessive viscosity caused by a high collodion concentration. In this embodiment, the mass concentration of the collodion solution is within the range of 3% to 5%, which is conducive to film formation.

[0042] Based on the above embodiment, as an alternative embodiment, the volume ratio of anhydrous ethanol to diethyl ether in the mixture of anhydrous ethanol and diethyl ether is 1:9 to 1:4. Preferably, the volume ratio of anhydrous ethanol to diethyl ether in the mixture of anhydrous ethanol and diethyl ether is 1:8. Thus, a volume ratio of anhydrous ethanol to diethyl ether within the above range can both dilute the collodion and prevent its precipitation.

[0043] Step S130: heating and stirring the pretreated porous resin and the coating solution at a preset temperature to immobilize the PEGylated uricase on the pretreated porous resin, thereby preparing a uric acid adsorbent.

[0044] Specifically, the pretreated porous resin is added to the coating solution in a mass ratio of 1:2 to 1:3, and the mixture is heated and stirred continuously in a constant temperature water bath at 45°C to 55°C until the solvent is completely evaporated, so that the PEGylated uricase is immobilized on the pretreated porous resin to prepare a uric acid adsorbent.

[0045] In this embodiment, the pretreated porous resin is added to the coating solution, allowing the pretreated porous resin to be soaked in the coating solution. The coating solution can coat the surface of the pretreated porous resin and enter the pores of the pretreated porous resin through the pores. By continuously heating and stirring in a constant temperature water bath at 45°C to 55°C, the coating solution forms a film layer on the surface and within the pores of the pretreated porous resin. PEGylated uricase is evenly distributed within this film layer, thereby achieving immobilization of the PEGylated uricase on the pretreated porous resin. By limiting the mass ratio of the pretreated porous resin to the coating solution within the above range, the coating solution can be ensured to fully soak the pretreated porous resin, forming a film layer on the surface and within the pores of the pretreated porous resin, while also avoiding waste of the coating solution.

[0046] Based on the above embodiment, as an optional embodiment, after immobilizing the PEGylated uricase on the pretreated porous resin and before preparing the uric acid adsorbent, the method further comprises:

[0047] The pretreated porous resin immobilized with PEGylated uricase is first rinsed with injection water, then drained, vacuum-dried, and cooled to room temperature in sequence to obtain a uric acid adsorbent. Thus, by cleaning the pretreated porous resin immobilized with PEGylated uricase, it is possible to avoid residual organic matter that affects the adsorption performance and safety performance of the uric acid adsorbent. As an optional embodiment, when vacuum drying the pretreated porous resin immobilized with PEGylated uricase, the pretreated porous resin immobilized with PEGylated uricase can be placed in a forced air oven, heated from room temperature to 50°C at a heating rate of 1°C / min to 5°C / min, and maintained at 50°C for 1 to 2 hours.

[0048] Compared with the prior art, the preparation method of the uric acid adsorbent provided in this embodiment uses a porous resin containing carboxyl groups as a carrier and carboxyl groups as a connecting structure. PEGylated uricase is chemically grafted to the surface of the porous resin via an esterification reaction, thereby immobilizing the PEGylated uricase on the surface of the pretreated porous resin. Subsequently, a coating solution is prepared using a PEGylated uricase solution and a collodion solution. The coating solution and the pretreated porous resin are heated and stirred to immobilize the PEGylated uricase on the pretreated porous resin. The PEGylated uricase can be immobilized on the surface and pores of the pretreated porous resin by physical encapsulation. Immobilization of the PEGylated uricase by physical encapsulation can prevent the shedding of particles and avoid the occurrence of thromboembolism. In addition, physical encapsulation with collodion, where the collodion is coated on the surface and pores of the porous resin, can improve the biocompatibility and safety of the porous resin and significantly enhance the overall performance of the uric acid adsorbent. In this embodiment, the combination of chemical grafting and physical encapsulation not only improves the firmness of the bond between the PEGylated uricase and the porous resin, but also allows the PEGylated uricase to be immobilized on the surface and within the pores of the porous resin. This fully utilizes the structure of the porous resin and greatly increases the immobilized capacity of the PEGylated uricase on the porous resin, enabling the uric acid adsorbent to accurately adsorb uric acid from the blood. Furthermore, the PEGylated uricase chemically grafted to the surface of the porous resin allows direct contact with the uric acid in the blood, degrading it. The physically encapsulated PEGylated uricase achieves a "sustained release" effect, extending the duration of action of the PEGylated uricase while adsorbing uric acid, thereby further degrading uric acid in the blood. The uric acid adsorbent provided in this embodiment has excellent adsorption properties for uric acid in the blood. Furthermore, the PEGylated uricase has advantages such as a long half-life and a low immune response, which can reduce the side effects and complications of existing uric acid removal methods. The uric acid adsorbent has good safety.

[0049] The second aspect of the embodiments of the present application provides a uric acid adsorbent, which is prepared using the preparation method described in the first aspect.

[0050] The uric acid adsorbent provided in this embodiment has good adsorption properties for uric acid and is safer than existing uric acid adsorption products.

[0051] A third aspect of the embodiments of the present application provides an application of a uric acid adsorbent, which can be used as an adsorption filler in a hemoperfusion device, a hemoadsorber, or a plasmaadsorber.

[0052] The uric acid adsorbent provided in this embodiment can be used in hemoperfusion devices, hemoadsorbers, or plasmaadsorbers, which can reduce uric acid in the blood. It is safer and can reduce the side effects and complications of uric acid removal methods.

[0053] In order to further explain the present invention in detail, the present invention will be further described below with reference to specific examples. Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods; unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention are all purchased from the market.

[0054] Example 1

[0055] This embodiment provides a method for preparing a uric acid adsorbent, comprising the following steps:

[0056] (1) 1 g of 95% pure PEGylated uricase lyophilized powder was dissolved in 500 mL of DMF (NN dimethylformamide) to obtain a first PEGylated uricase solution. 200 g of dried carboxyl-containing carbonized resin (commercially available carbonized resin with the designation SM-08-H, having a water content of less than 20 wt%) was added to the first PEGylated uricase solution. 1.2 g of dicyclohexylcarbodiimide (DCC) was slowly added, and 0.5 mL of 4-dimethylaminopyridine (DMAP) was slowly added dropwise in an ice bath. The mixture was reacted for 6 h to graft the PEGylated uricase onto the carbonized resin to obtain a pretreated carbonized resin. The pretreated carbonized resin was then washed with water and dried.

[0057] (2) 0.75 g of 95% pure PEGylated uricase lyophilized powder was dissolved in 250 mL of 0.2 mol / L Tris-HCl buffer solution at a pH of 7.4 to generate an intermediate solution with a mass concentration of 3 mg / mL; 100 mL of the intermediate solution was added to 100 mL of a mixture of anhydrous ethanol and water, wherein the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water was 50%, and stirred or shaken until completely dissolved to obtain a second PEGylated uricase solution, wherein the mass concentration of the second PEGylated uricase solution was 1.5 mg / mL.

[0058] (3) Take 30 g of 10% collodion and mix it evenly with 100 mL of anhydrous ethanol and 900 mL of ether solution to obtain a 3% by mass collodion solution.

[0059] (4) 200 mL of the second PEGylated uricase solution with a mass concentration of 1.5 mg / mL in step (2) and 200 mL of the 3% collodion solution in step (3) were mixed and stirred thoroughly to prepare a coating solution.

[0060] (5) Take 200 g of the pretreated carbonized resin in step (1) and add it to 400 mL of the coating solution for mixing and stirring. Adjust the temperature of the constant temperature water bath to no more than 55 ° C. By continuously heating and stirring, the solvent is completely evaporated until the carbonized resin is dry to obtain the coated carbonized resin. Then, the coated carbonized resin is rinsed with injection water until no foreign matter is visible to the naked eye, drained for 1 to 2 hours, placed in a vacuum chamber at 50 ° C. and dried for 2 hours. After cooling to room temperature, a uric acid adsorbent is obtained.

[0061] Example 2

[0062] This embodiment provides a method for preparing a uric acid adsorbent, comprising the following steps:

[0063] (1) 0.5 g of 95% pure PEGylated uricase lyophilized powder was dissolved in 500 mL of DMF (NN dimethylformamide) to obtain a first PEGylated uricase solution. 100 g of dried carboxyl-containing carbonized resin (commercially available carbonized resin with the designation SM-08-H, having a water content of less than 20 wt%) was added to the first PEGylated uricase solution. 0.6 g of dicyclohexylcarbodiimide (DCC) was slowly added. 0.25 mL of 4-dimethylaminopyridine (DMAP) was slowly added dropwise in an ice bath. The mixture was reacted for 4 h to graft the PEGylated uricase onto the carbonized resin to obtain a pretreated carbonized resin. The pretreated carbonized resin was then washed with water and dried.

[0064] (2) 1.25 g of high-purity PEGylated uricase lyophilized powder was dissolved in 250 mL of 0.2 mol / L Tris-HCl buffer solution at a pH of 7.4 to generate an intermediate solution with a mass concentration of 5 mg / mL; 100 mL of the intermediate solution was added to 25 mL of a mixture of anhydrous ethanol and water, wherein the volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water was 50%, and stirred or shaken until completely dissolved to obtain a second PEGylated uricase solution, wherein the mass concentration of the second PEGylated uricase solution was 4 mg / mL.

[0065] (3) Take 15 g of 10% collodion and mix it evenly with 50 mL of anhydrous ethanol and 250 mL of ether solution to obtain a 3% collodion solution.

[0066] (4) 100 mL of the second PEGylated uricase solution with a mass concentration of 4 mg / mL prepared in step (2) and 100 mL of the 5% collodion solution prepared in step (3) were mixed and stirred thoroughly to prepare a coating solution.

[0067] (5) Take 100 g of the pretreated carbonized resin in step (1) and add it to 200 mL of the coating solution for mixing and stirring. Adjust the temperature of the constant temperature water bath to no more than 55°C. By continuously heating and stirring, the solvent is completely evaporated until the resin is dry to obtain the coated carbonized resin. Then, the coated carbonized resin is rinsed with injection water until no foreign matter is visible to the naked eye, drained for 1 to 2 hours, placed in a vacuum drying oven below 50°C for 2 hours, and cooled to room temperature to obtain a uric acid adsorbent.

[0068] Test example

[0069] Clinically, hyperuricemia is defined as a uric acid concentration exceeding 420 μmol / L (7 mg / dL). In this test, a uric acid standard was purchased and the adsorption performance of the uric acid standard was tested using a static adsorption method. The specific test method is as follows:

[0070] Weigh 13.45 mg of uric acid standard powder (purchased from Beijing Laiyao Biological), dissolve it in 1 mL of 1 mol / L NaOH solution, and stir it with an oscillator until completely dissolved. Subsequently, take 0.5 mL of the above solution and add it to 49.5 mL of DMEM-F12 culture medium containing 10% FBS. Adjust the pH value to 7.2 to 7.4 with low concentration hydrochloric acid, and filter to obtain a uric acid solution with a final concentration of 0.8 mmol / L. Take another portion of the uric acid solution with a concentration of 0.8 mmol / L and dilute it to a concentration of 0.6 mmol / L.

[0071] Static adsorption was performed on the uric acid adsorbents prepared in Examples 1 and 2, respectively, at a volume ratio of 1:10. The initial uric acid concentrations were 0.6 mmol / L and 0.8 mmol / L, respectively. A blank solution was also prepared. The adsorption temperature was set to 37°C, and the adsorption was performed on a constant temperature shaker in a water bath at 140 rpm for 2 hours. Subsequently, the samples were removed and tested for uric acid concentration, yielding the test results shown in Table 1.

[0072] Table 1

[0073]

[0074] As can be seen from Table 1, the uric acid adsorbents prepared in Examples 1 and 2 have a significant adsorption effect on high-concentration uric acid, and the initial concentration of uric acid has little effect on the adsorption results, or basically no effect. This also shows that the use of the uric acid adsorbent provided by the present invention can achieve a rapid decrease in uric acid through the adsorption effect of the uric acid adsorbent, can significantly reduce uric acid in the blood, and the uric acid adsorbent is safer than existing uric acid adsorption products.

[0075] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for preparing a uric acid adsorbent, characterized in that: include: Grafting PEGylated uricase onto a porous resin containing carboxyl groups to prepare a pretreated porous resin; The second PEGylated uricase solution and the collodion solution are mixed evenly to prepare a coating solution; The pretreated porous resin and the coating solution are heated and stirred at a preset temperature to immobilize the PEGylated uricase on the pretreated porous resin, thereby preparing a uric acid adsorbent.

2. The method for preparing a uric acid adsorbent according to claim 1, wherein: The method of grafting PEGylated uricase onto a porous resin containing carboxyl groups to prepare a pretreated porous resin comprises: preparing the PEGylated uricase into a first PEGylated uricase solution; The carboxyl-containing porous resin and dicyclohexylcarbodiimide are added to the first PEGylated uricase solution, and then a 4-dimethylaminopyridine solution is added dropwise, and the reaction is carried out under an ice bath to graft the PEGylated uricase onto the porous resin, thereby preparing the pretreated porous resin.

3. The method for preparing a uric acid adsorbent according to claim 2, wherein: The first PEGylated uricase solution is prepared using an anhydrous DMF solution, wherein the mass concentration of the first PEGylated uricase solution is 1% to 3%; The amount of the dicyclohexylcarbodiimide used is 4‰ to 8‰ of the porous resin containing a carboxyl group.

4. The method for preparing a uric acid adsorbent according to claim 1, wherein: The second PEGylated uricase solution and the collodion solution are mixed evenly to prepare the coating solution, comprising: preparing the second PEGylated uricase solution; preparing the collodion solution; The second PEGylated uricase solution and the collodion solution are mixed in a volume ratio of 1:1 to 1:3, and stirred until the mixture is clear to prepare the coating solution. The volume content of the PEGylated uricase in the coating solution is 2.5‰ to 2%.

5. The method for preparing a uric acid adsorbent according to claim 4, wherein: The preparation of the second PEGylated uricase solution comprises: Dissolving PEGylated uricase in a buffer solution to prepare an intermediate solution; adding a mixture of anhydrous ethanol and water to the intermediate solution, stirring and mixing uniformly to prepare the second PEGylated uricase solution; The mass concentration of the intermediate solution is not higher than 5 mg / mL, and the mass concentration of the second PEGylated uricase solution is not lower than 1 mg / mL.

6. The method for preparing a uric acid adsorbent according to claim 5, characterized in that: The buffer solution is a trimethylammonium hydrochloride solution, the concentration of the trimethylammonium hydrochloride solution is 0.1 to 0.2 mol / L, and the pH value of the trimethylammonium hydrochloride solution is 7.2 to 7.4; The volume fraction of anhydrous ethanol in the mixture of anhydrous ethanol and water does not exceed 50%.

7. The method for preparing a uric acid adsorbent according to claim 4, wherein: The preparation of the collodion solution comprises: adding collodion to a mixture of anhydrous ethanol and ether, stirring and mixing uniformly to prepare the collodion solution; The volume ratio of the anhydrous ethanol to the ether is 1:9 to 1:4; and the mass concentration of the collodion solution is 3% to 5%.

8. The method for preparing a uric acid adsorbent according to claim 1, wherein: The step of heating and stirring the pretreated porous resin and the coating solution at a preset temperature to immobilize the PEGylated uricase on the pretreated porous resin comprises: The pretreated porous resin was added to the coating solution in a mass ratio of 1:2 to 1:3, and the mixture was heated and stirred in a constant temperature water bath at 45° C. to 55° C. until the solvent was completely evaporated, thereby immobilizing the PEGylated uricase on the pretreated porous resin.

9. A uric acid adsorbent, characterized in that: The method is as described in any one of claims 1 to 8.

10. An application of a uric acid adsorbent, characterized in that: The uric acid adsorbent prepared by the preparation method according to any one of claims 1 to 8, or the uric acid adsorbent according to claim 9, is used as an adsorption filler in a hemoperfusion device, a hemoadsorber, or a plasma adsorber.

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