WO2022147847A1 - Adsorbent for removing protein-bound uremic toxins by means of blood perfusion and preparation method therefor - Google Patents

Abstract

An adsorbent for removing protein-bound uremic toxins by means of blood perfusion and a preparation method therefor. The adsorbent for removing the protein-bound uremic toxins by means of blood perfusion is a porous resin having an amide group and taking polystyrene-acrylonitrile-divinylbenzene as a skeleton, wherein the porous resin has imprinted holes of imprinted molecules, and the imprinted molecules comprise protein-bound toxins and/or analogs thereof. The adsorbent has a significant removal effect on protein-bound toxins such as indoxyl sulfate and p-cresol sulfate, and also has a certain removal capacity on middle-macromolecule and small-molecule substances such as β2-microglobulin, vitamin B12, creatinine and pentobarbital sodium, and has excellent safety performance and mechanical strength.

Description

An adsorbent for removing protein-bound uremic toxins by blood perfusion and preparation method thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application requires the priority of the Chinese patent application with the application number 2021100063424 and the title of "An adsorbent for removing protein-bound uremic toxins from blood perfusion and its preparation method" filed with the China Patent Office on January 5, 2021 rights, the entire contents of which are incorporated herein by reference.

technical field

The present disclosure relates to the technical fields of chemical industry and biomedicine, in particular, to an adsorbent for removing protein-bound uremic toxins by blood perfusion and a preparation method thereof.

Background technique

Uremia is a clinical syndrome shared by various advanced chronic kidney disease (CKD), which refers to a comprehensive combination of a series of clinical manifestations that appear when chronic renal failure caused by CKD enters the end stage. sign. Uremic toxins refer to a large group of metabolic products that accumulate in the blood and tissues and are toxic when the renal function declines and the renal clearance rate of solutes decreases. The European Working Group on Uremic Toxins (EUTox) divides them into three categories according to their biochemical properties and removal methods: 1. Water-soluble, small molecular substances that are not bound to proteins, with a molecular mass usually less than 500, such as urea, creatinine, uric acid etc., such substances are easily removed by hemodialysis; 2. Medium molecular substances, the molecular weight is usually greater than 500, such as parathyroid hormone, β ₂ -microglobulin, leptin, etc., the conventional hemodialysis removal effect of such substances is not ideal , some substances can be removed by large-pore (high-flux) dialysis membranes and peritoneal dialysis; 3. Protein-bound toxoids, such as indoxyl sulfate, p-cresol sulfate, 3-carboxy-4-methyl-5- Propyl-2-furanpropionic acid (3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, CMPF), etc., most of the dialysis methods have poor removal effect on such substances.

Protein bound uremic toxins (PBUTs) are toxins that can reversibly bind to human serum albumin to form macromolecular complexes. There are currently 32 molecules known to be considered PBUTs, accounting for about 25% of uremic toxins, most of which are relatively small in molecular mass. PBUTs are mainly derived from protein degradation in food, and these degradation products are absorbed by intestinal epithelial cells and further metabolized by hepatocytes into the circulation. The binding of PBUTs to proteins leads to changes in the molecular structure, charge and even function of the proteins themselves. Relevant studies have shown that the free form of PBUTs, that is, not bound to proteins, is the main factor that causes tissue toxicity. Clinical studies have confirmed that with the renal clearance of uremic toxins in CKD patients significantly reduced, high concentrations of uremic toxins, especially PBUTs, are the primary cause of death and the main complication of cardiovascular disease (CVD) in CKD patients. important factor in development. In addition, a series of uremic complications such as immune dysfunction, organ fibrosis, central nervous system abnormalities, renal bone disease, and muscle weakness can also be caused by the accumulation of PBUTs in the body.

The more representative PBUTs are indoxyl sulfate (IS) and p-cresylsulphate (PCS), the relative molecular weights of which are 213.21 and 188.21 respectively, both of which belong to small molecular type organic compounds. Anionic toxins. They are all derived from the fermentative decomposition of dietary amino acids by intestinal bacteria, in which tryptophan is metabolized to indole, tyrosine and phenylalanine are metabolized to p-cresol, which is absorbed through the intestinal tract and then enters the liver through the portal vein. In the liver, IS and PCS are finally formed by sulfation, etc. It has been confirmed that in blood, the two compete with albumin for binding to the same site (sudlow site II) through non-covalent bonds such as electrostatic, dipole and van der Waals forces, and the binding rates are both above 90-95%. The protein binding constant measurement showed that the binding affinity of IS and PCS to albumin was moderate, and the binding was reversible, which was affected by blood temperature, pH value, dilution factor, and the concentration of ions and drugs in the blood. In vitro experiments and clinical studies have shown that both are significantly related to the incidence of chronic kidney disease, cardiovascular disease, and all-cause mortality. IS can cause a variety of pathological changes, such as promoting the occurrence of renal bone disease, causing vascular endothelial damage, etc. PCS can reduce the expression of endothelial cell adhesion molecules, which is related to the high incidence of infection and vascular calcification in maintenance hemodialysis (mainte-nance hemodialysis, MHD).

At present, MHD is the main alternative to prolong the life of patients with uremia, but clinical studies have shown that conventional low-flux hemodialysis (LFHD) treatment can effectively remove small molecule toxins from the body, and high-flux hemodialysis (high-flux hemodialysis) , HFHD) and peritoneal dialysis (PD) also have a certain removal effect on most medium and large molecular toxins, but PBUTs represented by IS and PCS cannot be carried out due to their binding to proteins and multi-chamber distribution. Effective clearance, it is reported that the clinical clearance rate of IS and PCS is about 30% for LFHD and less than 35% for HFHD, and the weekly clearance efficiency is less than 1/10 of that of the kidney, which will lead to the accumulation of these substances in the body and cause related Complications, affecting the therapeutic effect of hemodialysis, thereby affecting the quality of life of MHD patients.

In order to effectively remove PBUTs such as IS and PCS, other blood purification methods have been clinically developed and adopted. The common ones are: 1. Super-flux hemodialysis (SFHD) that increases the dialysis membrane pore size and ultrafiltration coefficient , can improve the clearance rate of IS, etc., but the patient loses more albumin; 2. Hemodiafiltration (HDF) formed by combining the diffusion mechanism of hemodialysis and the convection mechanism of hemofiltration can significantly improve the The clearance rate of medium and macromolecular toxins, but the relevant reports on the clearance effect of PBUTs are inconsistent, there is controversy, the clearance rate of IS and PCS is not more than 45% as a whole; HP), due to the low clearance rate of single HP to small molecule toxins such as urea and creatinine, and its own lack of ability to correct water, electrolyte and acid-base balance disorders, it is often combined with conventional HD to achieve complementary advantages. Clinical studies have shown that HP combined with HD can significantly improve the clearance of protein-bound toxoids. The clearance rate of IS and PCS can reach 50% to 60%, which is better than other blood purification methods, especially for CMPF with stronger binding force. The advantages of PBUTs are more obvious, and long-term HD+HP treatment can maintain a low level of protein-bound toxoids in MHD patients, and can improve the quality of life of MHD patients.

HP is a blood purification technology that introduces the patient's blood into a perfusion device equipped with solid adsorbents, and removes exogenous or endogenous toxins, drugs or metabolic wastes that cannot be removed by dialysis in the blood through adsorption. It is the earliest adsorption mode used for clinical removal of various uremic toxins. In addition to uremia, HP technology has been clinically applied to hyperbilirubinemia, acute poisoning, sepsis, hyperlipidemia, systemic Treatment of lupus erythematosus, myasthenia gravis, etc. The solid adsorbent in HP may be in the form of spherical particles, fiber bundles or membranes, and its performance is affected by the specific surface area of the adsorbent, relative molecular weight of the solute, molecular structure, temperature, pH value, etc., and is most commonly used in the treatment of uremia patients. The adsorbent materials are activated carbon and resin.

At present, there have been many clinical reports on the removal of PBUTs by HD+HP. The hemoperfusion devices used, such as Zhuhai Jianfan HA series and Foshan Boxin MG series, are domestic products, and the adsorption material is macroporous adsorption resin. HP can effectively remove PBUTs such as IS, PCS, CMPF, hippuric acid (HA), homocysteine (Hcy), and advanced glycation end products (AGEs). , The overall clearance rate of HA130 hemoperfusion device combined with HD on IS and PCS is about 50%, and MG150 combined with HD is about 55%, which is significantly better than HD and slightly better than HDF. In addition, the clearance rate of small molecule toxins such as blood urea nitrogen (BUN) and serum creatinine (sCr) by HA130 combined with HD is about 60%, which is comparable to HD; (intact parathyroid hormone, iPTH), MG350 combined with HD have a clearance rate of about 40% for medium molecular toxins such as β2-microglobulin (β2-MG), which is significantly better than HD, but slightly lower than HDF. It can be seen that although the above products have achieved good clinical therapeutic effects, there is still room for improvement in continuing to improve the PBUTs scavenging ability and simultaneously removing medium molecular toxins.

With this in mind, the present disclosure is hereby made.

SUMMARY OF THE INVENTION

The objectives of the present disclosure include, for example, to provide an adsorbent for removing protein-bound uremic toxins by blood perfusion, which has a significant removal effect on protein-bound toxoids such as indoxyl _sulfate , p-cresol sulfate, etc. - Microglobulin, vitamin B ₁₂ , creatinine, pentobarbital sodium and other medium and large molecules and small molecules also have certain scavenging ability, and have excellent safety performance and mechanical strength.

The purpose of the present disclosure also includes, for example, to provide a preparation method of an adsorbent for removing protein-bound uremic toxins by blood perfusion, the method has simple steps, mild conditions, is conducive to environmental protection and cost reduction, and the obtained adsorbent is in While maintaining good adsorption performance, it has high mechanical strength and good adsorption kinetics.

In order to achieve the above-mentioned purpose of the present disclosure, the following technical solutions are specially adopted:

An adsorbent for removing protein-bound uremic toxins by blood perfusion, which is a porous resin with an amide group and taking polystyrene-acrylonitrile-divinylbenzene as a skeleton, and the porous resin has imprinting of imprinted molecules Cavity; the imprinted molecule includes protein-binding toxoids and/or analogs of protein-binding toxoids.

In one or more embodiments, the content of amide groups in the adsorbent is 1.0-2.5 mmol/g dry resin;

In one or more embodiments, the particle size of the adsorbent is 0.4-1.2 mm;

In one or more embodiments, the moisture content of the adsorbent is 50% to 70%;

In one or more embodiments, the specific surface area of the adsorbent is 700-900 m ² /g;

In one or more embodiments, the sphericity after grinding of the adsorbent is ≥90%;

In one or more embodiments, the protein-bound toxoid comprises at least one of p-cresol sulfate and indoxyl sulfate;

In one or more embodiments, the analog of the protein-bound toxoid includes at least one of p-toluenesulfonic acid and L-tryptophan.

The above-mentioned preparation method of the adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) the oil phase is mixed with the water phase to carry out a polymerization reaction, and resin A is obtained after the separation;

the oil phase includes styrene, acrylonitrile, divinylbenzene, porogen and benzoyl peroxide;

the aqueous phase includes gelatin, sodium chloride and water;

(b) in the resin A that step (a) obtains, add the vitriol oil to carry out hydrolysis reaction, obtain resin B;

(c) mixing the ethanol solution of the imprinted molecule, the resin B, 1,2-dichloroethane and anhydrous ferric chloride to carry out a cross-linking-imprinting reaction to obtain a protein-bound uremic drug for blood perfusion removal Adsorbents for toxins.

In one or more embodiments, in step (a), the oil phase is mainly composed of the following components in parts by mass: 5-15 parts of styrene, 5-15 parts of acrylonitrile, 80% diethylene 70-90 parts of benzene, 100-150 parts of porogen and 0.5-1.5 parts of benzoyl peroxide;

In one or more embodiments, the aqueous phase includes the following components in mass concentration: gelatin 0.5%-2% and sodium chloride 5%-10%;

In one or more embodiments, the mass ratio of the water phase to the oil phase is (2.5-4):1;

In one or more embodiments, the mass ratio of the imprinted molecules to the resin B is (1-4):20;

In one or more embodiments, the mass ratio of the anhydrous ferric chloride to the resin B is (3-8):20;

In one or more embodiments, the porogen includes component A and component B, the component A is selected from alkanes and/or aromatic hydrocarbons, and the component B is selected from alcohols and/or esters;

In one or more embodiments, the component A is selected from at least one of toluene, ethylbenzene, xylene, n-heptane and 200# gasoline;

In one or more embodiments, the component B is selected from at least one of cyclohexanol, isoamyl alcohol, n-octanol, dodecanol and butyl acetate;

In one or more embodiments, the mass of the component A is 50% to 70% of the total mass of the porogen.

In one or more embodiments, in step (a), the initial mixing temperature of the oil phase and the water phase is 48-52°C;

In one or more embodiments, the oil phase and the water phase are mixed and left to stand, followed by stirring, heating and heat preservation;

In one or more embodiments, the standing time is 8-12 min;

In one or more embodiments, the heating is to raise the temperature to 78-90°C at a rate of 0.8-1.1°C/2min;

In one or more embodiments, the incubation is performed at 78-90° C. for 4-12 h.

In one or more embodiments, in step (a), the separation comprises: performing solid-liquid separation on the reacted mixture, and performing water washing, alcohol washing and sieving on the obtained resin to obtain resin A;

In one or more embodiments, the temperature of the water washing is 48-52°C.

In one or more embodiments, in step (b), the concentrated sulfuric acid is slowly added to the resin A at 20-25° C. and stirred, and after the solid-liquid separation of the reacted mixture, gradient concentrated sulfuric acid is used. The separated resin was washed with water until neutral, and dried to obtain resin B.

In one or more embodiments, the time for slowly adding the concentrated sulfuric acid to the resin A at 20-25° C. and stirring for 8-12 hours;

In one or more embodiments, the concentration of the concentrated sulfuric acid is 90% to 95%;

In one or more embodiments, the drying temperature for obtaining resin B after drying is 70-78°C;

In one or more embodiments, the resin B is dried to less than 2% moisture.

In one or more embodiments, in step (c), the mixture of the ethanol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is stirred and swollen, and then anhydrous trichloride is added. The iron is heated, heated and kept warm, and the solid-liquid is separated to obtain an adsorbent for blood perfusion to remove protein-bound uremic toxins;

In one or more embodiments, the temperature for stirring the mixture of the ethanol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is 28-32° C., and the time is 1.8-2.2 h ;

In one or more embodiments, the amount of the resin B is 20 parts by mass;

In one or more embodiments, the alcoholic solution of the imprinted molecule is composed of: 1-4 parts by mass of the imprinted molecule and 10-20 parts by volume of anhydrous ethanol;

In one or more embodiments, the amount of the 1,2-dichloroethane is 80-120 parts by mass;

In one or more embodiments, the heating is increased to 65-80° C., and the holding time is 8-16 h;

In one or more embodiments, the amount of the anhydrous ferric chloride is 3-8 parts by mass.

In one or more embodiments, the resin after the solid-liquid separation described in step (c) is washed with ethanol and washed with water, the resin is packed into a column, washed with an acetone-acid solution, washed with water until neutral, and dried;

In one or more embodiments, the number of times of washing with ethanol is 2 to 3 times, and the number of times of washing with water is 2 to 3 times; the amount of each time is 180 to 220 parts by mass;

In one or more embodiments, the acetone-acid solution is composed of acetone, water and hydrochloric acid in a volume ratio of (4.9-5.1):(3.9-4.1):1;

In one or more embodiments, the amount of the acetone-acid solution is 8-12 bed volumes.

Compared with the prior art, the beneficial effects of the present disclosure are:

(1) The hemoperfusion adsorbent of the present disclosure adopts ternary copolymerization to introduce polar groups, and combines with the imprinted template molecules introduced on the basis of the post-crosslinking reaction of dangling double bonds. The matching molecularly imprinted pore structure such as p-cresol sulfate can selectively improve the removal of free PBUTs, and the effect is obvious. In addition, the residual amide group on the resin enhances the hydrophilicity and biocompatibility of the adsorbent.

(2) In the hemoperfusion adsorbent provided by the present disclosure, a mesoporous structure of 20-50 nm is introduced through the use of a mixed porogen in the suspension polymerization process, and a microporous structure of less than 20 nm is introduced into the cross-linking reaction after dangling double bonds, so that the adsorption While removing PBUTs, the agent also maintains a certain ability to remove medium-sized and small-molecule toxins. Moreover, the multi-component pore structure enables the adsorbent to maintain good adsorption performance while maintaining high mechanical strength and good adsorption kinetics. The introduction of capillary pores also improves the surface hydrophilicity and hydrophobicity and blood compatibility of the adsorbent. Compared with commercially available post-crosslinking macroporous adsorbents, the post-crosslinking reaction of pendant double bonds has simple steps, mild conditions, and reduces the use of a large amount of organic solvents, which is beneficial to environmental protection and cost reduction.

Detailed ways

The embodiments of the present disclosure will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

According to one aspect of the present disclosure, the present disclosure relates to an adsorbent for hemoperfusion to remove protein-bound uremic toxins, which is a porous resin with amide groups and with polystyrene-acrylonitrile-divinylbenzene as a skeleton , the porous resin has imprinted cavities of imprinted molecules; the imprinted molecules include protein-binding toxoids and/or analogs of protein-binding toxoids.

The adsorbent of the present disclosure has a significant scavenging effect on protein-bound toxoids such as indoxyl sulfate and p-cresol sulfate, and has a significant scavenging effect on medium and large molecules such as β ₂ -microglobulin, vitamin B ₁₂ , creatinine, sodium pentobarbital, and the like. Small molecular substances also have a certain scavenging ability, and have good safety performance and mechanical strength.

Preferably, the content of amide groups in the adsorbent is 1.0-2.5 mmol/g dry resin.

In one embodiment, the content of amide groups in the adsorbent is 1.0-2.5 mmol/g, and 1.0 mmol/g, 1.1 mmol/g, 1.2 mmol/g, 1.3 mmol/g, 1.4 mmol/g can also be selected. g, 1.5mmol/g, 1.6mmol/g, 1.7mmol/g, 1.8mmol/g, 1.9mmol/g, 2mmol/g, 2.1mmol/g, 2.2mmol/g, 2.3mmol/g, 2.4mmol/g or 2.5mmol/g.

Preferably, the particle size of the adsorbent is 0.4-1.2 mm.

In one embodiment, the particle size of the adsorbent is 0.4-1.2 mm, and can also be selected from 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm or 1.2 mm.

Preferably, the moisture content of the adsorbent is 50% to 70%.

In one embodiment, the moisture content of the adsorbent is 50% to 70%, and 50%, 52%, 55%, 57%, 60%, 62%, 65% or 70% can also be selected.

Preferably, the specific surface area of the adsorbent is 700-900 m ² /g.

In one embodiment, the specific surface area of the adsorbent is 700-900 m ² /g, and 700 m ² /g, 710 m ² /g, 720 m ² /g, 730 m ² /g, 740 m ² /g, 750m ² /g, 760m ^{2 /g, 770m 2 /} g, 780m ² /g, 790m ² /g, 800m ² /g, 810m ² /g, 820m ^{2 /g, 830m 2 /} g, 840m ² /g, 850m ² /g, 860m ² /g, 870m ² /g, 880m ² /g, 890m ² /g or 900m ² /g.

Preferably, the sphericity of the adsorbent after grinding is ≥90%.

In one embodiment, the sphericity after grinding of the adsorbent is ≥90%, and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% can also be selected .

Preferably, the protein-bound toxoid comprises at least one of p-cresol sulfate and indoxyl sulfate.

Preferably, the analog of the protein-bound toxoid includes at least one of p-toluenesulfonic acid and L-tryptophan.

According to another aspect of the present disclosure, the present disclosure also relates to the above-mentioned preparation method of the adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) the oil phase is mixed with the water phase to carry out a polymerization reaction, and resin A is obtained after the separation;

the oil phase includes styrene, acrylonitrile, divinylbenzene, porogen and benzoyl peroxide;

the aqueous phase includes gelatin, sodium chloride and water;

(b) in the resin A that step (a) obtains, add the vitriol oil to carry out hydrolysis reaction, obtain resin B;

(c) mixing the ethanol solution of the imprinted molecule, the resin B, 1,2-dichloroethane and anhydrous ferric chloride to carry out a cross-linking-imprinting reaction to obtain a protein-bound uremic drug for blood perfusion removal Adsorbents for toxins.

The preparation method of the present disclosure has simple steps, mild conditions, and reduces the use of a large amount of organic solvents, which is beneficial to environmental protection and cost reduction.

Step (a) of the preparation method of the present disclosure is normal-phase suspension polymerization to obtain porous white spheres with polystyrene-acrylonitrile-divinylbenzene skeleton; step (b) hydrolyzes the obtained porous white spheres at room temperature with concentrated sulfuric acid to obtain amide group-containing white spheres The adsorption resin; step (c) suspending the double bond after the cross-linking reaction, and adding protein-bound toxoid or a small molecular substance with similar structure as an imprinting template during the reaction, forming a molecularly imprinted cavity, micropores and intermediates at the same time. Pore-structured novel hemoperfusion sorbent for the removal of protein-bound uremic toxins.

The polymerization process makes the adsorbent have a mesoporous structure of 20-50nm through a single or mixed porogen, which is conducive to the removal of medium and large molecular toxins with a molecular weight of 5000-20000, and uses ternary copolymerization to introduce polar molecules that can interact with imprinted template molecules. sex group. The post-crosslinking reaction of hanging double bonds can increase the microporous structure below 20nm and improve the removal of small molecule toxins with molecular weight below 500. The imprinting template molecules introduced on this basis can selectively improve the ability of free protein-bound uremic toxins. adsorption.

Preferably, the oil phase is mainly composed of the following components in parts by mass: 5-15 parts of styrene, 5-15 parts of acrylonitrile, 70-90 parts of 80% divinylbenzene, 100-150 parts of porogen and 0.5-1.5 parts of benzoyl peroxide.

In one embodiment, the styrene is 5-15 parts, and 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts or 15 parts can also be selected. share.

In one embodiment, the acrylonitrile is 5 to 15 parts, and 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts or 15 parts can also be selected. share.

In one embodiment, the 80% divinylbenzene is 70 to 90 parts, and 70 parts, 75 parts, 80 parts, 85 parts or 90 parts can also be selected.

In one embodiment, 100 to 150 parts of the porogen, 100 parts, 110 parts, 120 parts, 130 parts, 140 parts or 150 parts can also be selected.

Preferably, the porogen includes component A and component B, the component A is selected from alkanes and/or aromatic hydrocarbons, and the component B is selected from alcohols and/or esters.

Preferably, the component A is selected from at least one of toluene, ethylbenzene, xylene, n-heptane and 200# gasoline.

Preferably, the component B is selected from at least one of cyclohexanol, isoamyl alcohol, n-octanol, dodecanol and butyl acetate.

Preferably, the mass of the component A is 50% to 70% of the total mass of the porogen.

In one embodiment, the benzoyl peroxide is 0.5 to 1.5 parts, and 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 part, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts can also be selected. servings or 1.5 servings.

Preferably, the aqueous phase includes the following components in mass concentration: 0.5%-2% of gelatin and 5%-10% of sodium chloride.

Preferably, the mass ratio of the water phase to the oil phase is (2.5-4):1.

In one embodiment, the mass ratio of the water phase to the oil phase is (2.5-4):1, and 2.5:1, 3:1, 3.5:1 or 4:1 can also be selected.

Preferably, in step (a), the initial mixing temperature of the oil phase and the water phase is 48-52°C.

In one embodiment, in step (a), the initial mixing temperature of the oil phase and the water phase is 48-52°C, and 48°C, 49°C, 50°C, 51°C or 52°C can also be selected .

Preferably, the oil phase and the water phase are mixed and left to stand, followed by stirring, heating and heat preservation.

Preferably, the standing time is 8-12 min.

In one embodiment, the standing time is 8-12 minutes, and 8 minutes, 9 minutes, 10 minutes, 11 minutes or 12 minutes can also be selected.

Preferably, the heating is to raise the temperature to 78-90°C at a rate of 0.8-1.1°C/2min.

In one embodiment, the heating is to raise the temperature to 78-90°C at a rate of 1°C/2min.

Preferably, the temperature is kept at 78-90° C. for 4-12 hours.

Preferably, in step (a), the separation comprises: performing solid-liquid separation on the reacted mixture, and performing water washing, alcohol washing and sieving on the obtained resin to obtain resin A.

Preferably, the temperature of the water washing is 48-52°C.

In one embodiment, the temperature of the water washing is 48-52°C, and 48°C, 49°C, 50°C, 51°C or 52°C can also be selected.

Preferably, in step (b), the concentrated sulfuric acid is slowly added to the resin A at 20-25° C. and stirred, and after the solid-liquid separation of the reacted mixture, gradient concentrated sulfuric acid is used to wash the separated resin , washed with water until neutral, and dried to obtain resin B.

Preferably, the time for adding the concentrated sulfuric acid at 20-25° C. and stirring is 8-12 h.

Preferably, the concentration of the concentrated sulfuric acid is 90% to 95%.

Preferably, the drying temperature for obtaining resin B after drying is 70-78°C.

In one embodiment, the drying temperature of resin B obtained after drying is 70-78°C, and 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C can also be selected. or 78°C.

Preferably, the resin B is dried to a moisture content of less than 2%.

Preferably, in step (c), the mixture of the ethanol alcohol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is stirred and swollen, and then anhydrous ferric chloride is added and heated and heated. Heat preservation and solid-liquid separation to obtain an adsorbent for blood perfusion to remove protein-bound uremic toxins.

Preferably, the mass ratio of the 1,2-dichloroethane to the resin B is (80-120):20.

Preferably, the mass ratio of the imprinted molecules to the resin B is (1-4):20.

Preferably, the mass/volume ratio of the imprinted molecule to the absolute ethanol solvent is (1-4):(10-20).

Preferably, the mass ratio of the anhydrous ferric chloride to the resin B is (3-8):20.

Preferably, the temperature for stirring the mixture of the ethanol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is 28-32° C., and the time is 1.8-2.2 h.

In one embodiment, the temperature at which the mixture of the ethanol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is stirred is 28-32° C., and 28° C., 29° C., 30°C, 31°C or 32°C.

In one embodiment, the time is 1.8-2.2h, and 1.8h, 1.9h, 2h, 2.1h or 2.2h can also be selected.

Preferably, the heating temperature is increased to 65-80° C., and the holding time is 8-16 h.

Preferably, the amount of the resin B used is 20 parts by mass.

Preferably, the alcohol solution of the imprinted molecules is composed of: 1-4 parts by mass of the imprinted molecules and 10-20 parts by volume of absolute ethanol.

Preferably, the amount of the 1,2-dichloroethane is 80-120 parts by mass.

Preferably, the resin after the solid-liquid separation in step (c) is washed with ethanol and water, then washed with acetone-acid solution, washed with water until neutral, and dried.

Preferably, the ethanol washing times are 2-3 times, and the water washing times are 2-3 times; the dosage for each time is 180-220 parts by mass.

Preferably, the acetone-acid solution is composed of acetone, water and hydrochloric acid in a volume ratio of (4.9-5.1):(3.9-4.1):1.

Preferably, the dosage of the acetone-acid solution is 8-12 bed volumes.

In one embodiment, the volume ratio of the acetone, water and hydrochloric acid is 5:4:1; the consumption of the acetone-acid solution is 10 bed volumes.

The present disclosure will be further explained below with reference to specific embodiments.

Example 1

A preparation method of an adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) Polymerization reaction: 15 parts by mass of styrene, 15 parts by mass of acrylonitrile, 70 parts by mass of 80% divinylbenzene, 50 parts by mass of toluene, 50 parts by mass of n-octanol, 1.5 parts by mass of Benzoyl oxide (BPO) is evenly mixed and prepared into an oil phase, and put into a pre-prepared water phase containing 1% mass concentration of gelatin and 10% mass concentration of sodium chloride at 50 ° C. The quality of the water phase and the oil phase is The ratio is 4:1; after mixing, let stand for 10 minutes, start stirring, adjust the particle size to an appropriate range, then heat up to 90 °C at a rate of 1 °C/2min, and keep it for 12 h; stop the reaction, drain the polymerization mother liquor, and wash with water at 50 °C The resin is clarified to the effluent, the porogen is extracted with ethanol at room temperature, and then transferred to the water phase, and sieved in the wet state to obtain 0.4-1.2 mm resin, and the free water is drained for use;

(b) hydrolysis reaction: slowly add 95% concentrated sulfuric acid to the resin obtained in the above step (a), and under stirring at 25 ° C, react for 12 hours; stop the reaction, drain the hydrolysis mother liquor, and use a gradient of sulfuric acid with a concentration from high to low. The resin is washed until the water is neutral; the free water is drained, the resin is air-dried, and then dried in an oven at 75°C until the moisture is less than 2%, for use;

(c) Post-crosslinking-imprinting reaction: 80 parts by volume of 1,2-dichloroethane was added to 20 parts by mass of the resin obtained in the above step (b), and then 3 parts by mass of indoxyl sulfate pre-dissolved was added 20 parts by volume of anhydrous ethanol, swelled under stirring at 30°C for 2h, added 4 parts by mass of anhydrous ferric chloride, and then heated to 65°C for 10h reaction; stopped the reaction, drained the reaction mother liquor, and added ethanol and water in turn. The resin was washed 3 times (200 parts by mass each time), and then the resin was packed into a column, washed with acetone-acid solution (acetone: water: hydrochloric acid = 5:4:1, volume ratio) for 10 BV, and washed with pure water to neutrality; The free water was dried to obtain the finished product.

Example 2

A preparation method of an adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) Polymerization reaction: 5 parts by mass of styrene, 5 parts by mass of acrylonitrile, 90 parts by mass of 80% divinylbenzene, 100 parts by mass of toluene, 50 parts by mass of n-octanol, and 0.5 parts by mass of BPO Mix and evenly prepare into oil phase, put into pre-prepared water phase containing 0.5% mass concentration of gelatin and 5% mass concentration of sodium chloride at 50°C, and the mass ratio of water phase to oil phase is 3:1; After mixing, let it stand for 10 minutes, start stirring, adjust the particle size to an appropriate range, heat up to 80°C at a rate of 1°C/2min, and keep it for 10 hours; stop the reaction, drain the polymerization mother liquor, and wash the resin at 50°C until the effluent is clear. At room temperature, the porogen is extracted with ethanol, and then transferred to the water phase, sieved in the wet state to obtain 0.4-1.2 mm resin, and the free water is drained for use;

(b) hydrolysis reaction: slowly add 90% concentrated sulfuric acid to the resin obtained in the above step (a), and under stirring at 25° C., react for 8 hours; stop the reaction, drain the hydrolysis mother liquor, and use a gradient of sulfuric acid with a concentration from high to low. The resin is washed until the water is neutral; the free water is drained, the resin is air-dried, and then dried in an oven at 75°C until the moisture is less than 2%, for use;

(c) Post-crosslinking-imprinting reaction: 100 parts by volume of 1,2-dichloroethane was added to 20 parts by mass of the resin obtained in the above step (b), and then 2 parts by mass of indoxyl sulfate pre-dissolved was added 10 parts by volume of anhydrous ethanol, swelled under stirring at 30°C for 2h, added 6 parts by mass of anhydrous ferric chloride, and then heated to 80°C for 14h reaction; stopped the reaction, drained the reaction mother liquor, and added ethanol and water in turn. Wash the resin 3 times (200 parts by mass each time), then pack the resin into a column, wash 10BV with pyruvic acid solution (acetone:water:hydrochloric acid=5:4:1, volume ratio), then wash with pure water to neutrality; free water to obtain the finished product.

Example 3

A preparation method of an adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) Polymerization reaction: 5 parts by mass of styrene, 15 parts by mass of acrylonitrile, 80 parts by mass of 80% divinylbenzene, 100 parts by mass of n-heptane, 50 parts by mass of butyl acetate, 0.5 part by mass The BPO is mixed and evenly prepared into an oil phase, and put into a pre-prepared water phase containing 1% mass concentration of gelatin and 10% mass concentration of sodium chloride at 50°C, and the mass ratio of the water phase to the oil phase is 4:1 ; After mixing, let stand for 10 min, start stirring, adjust the particle size to an appropriate range, heat up to 80 °C at a rate of 1 °C/2min, and keep it for 8 hours; stop the reaction, drain the polymerization mother liquor, and wash the resin at 50 °C until the effluent is clear. , at room temperature, the porogen is extracted with ethanol and transferred to the water phase, and sieved in the wet state to obtain 0.4-1.2 mm resin, and the free water is drained for use;

(b) hydrolysis reaction: slowly add 95% concentrated sulfuric acid to the resin obtained in the above step (a), and under stirring at 20° C., react for 12 hours; stop the reaction, drain the hydrolysis mother liquor, and use a gradient of sulfuric acid with a concentration from high to low. The resin is washed until the water is neutral; the free water is drained, the resin is air-dried, and then dried in an oven at 75°C until the moisture is less than 2%, for use;

(c) Post-crosslinking-imprinting reaction: 120 parts by volume of 1,2-dichloroethane was added to 20 parts by mass of the resin obtained in the above step (b), and then 3 parts by mass of p-cresol sulfuric acid pre-dissolved was added 15 parts by volume of anhydrous ethanol, swelled under stirring at 30°C for 2h, added 8 parts by mass of anhydrous ferric chloride, and then heated to 80°C for 16h reaction; stopped the reaction, drained the reaction mother liquor, and added ethanol and water in turn. Wash the resin 3 times (200 parts by mass each time), then pack the resin into a column, wash 10BV with pyruvic acid solution (acetone:water:hydrochloric acid=5:4:1, volume ratio), then wash with pure water to neutrality; free water to obtain the finished product.

Example 4

A preparation method of an adsorbent for removing protein-bound uremic toxins by blood perfusion, comprising the following steps:

(a) Polymerization reaction: 5 parts by mass of styrene, 10 parts by mass of acrylonitrile, 85 parts by mass of 80% divinylbenzene, 100 parts by mass of toluene, 40 parts by mass of dodecanol, and 0.7 parts by mass of BPO Mix and evenly prepare the oil phase, put it into the pre-prepared water phase containing 0.7% mass concentration of gelatin and 5% mass concentration sodium chloride at 50°C, and the mass ratio of the water phase to the oil phase is 4:1; After mixing, let it stand for 10 minutes, start stirring, adjust the particle size to an appropriate range, heat up to 85°C at a rate of 1°C/2min, and keep it for 10 hours; stop the reaction, drain the polymerization mother liquor, and wash the resin at 50°C until the effluent is clear. At room temperature, the porogen is extracted with ethanol, and then transferred to the water phase, sieved in the wet state to obtain 0.4-1.2 mm resin, and the free water is drained for use;

(b) hydrolysis reaction: slowly add 90% concentrated sulfuric acid to the resin obtained in the above step (a), and under stirring at 25 ° C, react for 10 hours; stop the reaction, drain the hydrolysis mother liquor, and use a gradient of sulfuric acid with a concentration from high to low. The resin is washed until the water is neutral; the free water is drained, the resin is air-dried, and then dried in an oven at 75°C until the moisture is less than 2%, for use;

(c) Post-crosslinking-imprinting reaction: 120 parts by volume of 1,2-dichloroethane was added to 20 parts by mass of the resin obtained in the above step (b), and then 2 parts by mass of p-toluene dissolved in advance was added Sulfonic acid and 2 parts by mass of L-tryptophan in 20 parts by volume of anhydrous ethanol were swollen under stirring at 30°C for 2h, then 6 parts by mass of anhydrous ferric chloride was added, and the temperature was raised to 70°C for reaction for 14h; the reaction was stopped, The reaction mother liquor was drained, and the resin was washed 3 times with ethanol and water in turn (200 parts by mass each time), and then the resin was packed into a column and washed with pyruvic acid solution (acetone:water:hydrochloric acid=5:4:1, volume ratio) After 10BV, pure water is washed until neutral; free water is drained to obtain finished product.

Experimental example

1. The basic indicators of the adsorbent obtained in each embodiment of the present disclosure are shown in Table 1;

Table 1 Basic indicators of adsorbents

The adsorbent prepared by the method of the present disclosure is brown-yellow to brown-black opaque beads in appearance, with a particle size of 0.4-1.2 mm, a water content of 50-70%, and an amide group content of 1.0-2.5 mmol/g dry resin. The specific surface area is 700～900m ² /g, and the spherical rate after grinding is ≥90%.

2. The in vitro adsorption performance and safety performance of the hemoperfusion adsorbent obtained in each example after being coated with collodion, compared with the performance of the adsorbent in the commercially available perfusion device, the results are shown in Table 2, wherein indoxyl sulfate , p-cresol sulfate and β ₂ -microglobulin are absorbed by human blood, and vitamin B ₁₂ , creatinine and sodium pentobarbital are absorbed by standard solution.

Table 2 Adsorption performance and safety performance test results

In the present disclosure, the degree of crosslinking in the polymerization reaction, the amount and proportion of the porogen, the amount of acrylonitrile, the amount of benzoyl peroxide, the temperature and time of the polymerization reaction, the temperature and time of the post-crosslinking reaction, anhydrous Factors such as the dosage of ferric chloride, the dosage of imprinted template molecules and the ratio have obvious effects on the structure and adsorption performance of the adsorbent.

In the embodiment of the present disclosure, the removal rate of indoxyl sulfate by the adsorbent is ≥55%, the removal rate of p-cresol sulfate is ≥60%, the removal rate of β ₂ -microglobulin is ≥82%, and the removal of vitamin B ₁₂ (simulated middle molecule) The removal rate is ≥95%, the removal rate of creatinine is ≥65%, and the removal rate of sodium pentobarbital (simulated small molecule) is ≥98%. Red blood cells, white blood cells, platelets decreased rate of ≤ 10%, total protein adsorption rate ≤ 15%.

The adsorbent of the present disclosure has a significantly higher removal rate of indoxyl sulfate and p-cresol sulfate than the adsorbents used in the commercially available HA series and MG series hemoperfusion devices, and the removal rate of β ₂ -microglobulin is slightly higher than that of the HA adsorbent , creatinine removal rate was significantly higher than that of MG adsorbent.

Through molecular imprinting technology, the scavenging ability of the adsorbent for indoxyl sulfate and p-cresol sulfate was greatly improved, which was significantly higher than that of the commercial products. Thereby, it is further adsorbed by the microporous part of the adsorbent.

By adding a suitable pore-forming agent and controlling the degree of post-crosslinking in the first polymerization, the adsorbent has a suitable mesoporous structure and a large number of microporous structures, which can simultaneously maintain the resistance to medium and large molecules and small molecule toxins. Good removal effect, better than some commercially available products.

The in vitro safety performance is comparable to that of commercial products. The next step can be considered to optimize and enlarge the adsorbent for the investigation of animal experiments and clinical trials.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, but not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements to some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present disclosure. range.

Industrial Applicability

(1) The hemoperfusion adsorbent of the present disclosure adopts ternary copolymerization to introduce polar groups, and combines with the imprinted template molecules introduced on the basis of the post-crosslinking reaction of dangling double bonds. The matching molecularly imprinted pore structure such as p-cresol sulfate can selectively improve the removal of free PBUTs, and the effect is obvious. In addition, the residual amide group on the resin enhances the hydrophilicity and biocompatibility of the adsorbent.

(2) In the hemoperfusion adsorbent provided by the present disclosure, a mesoporous structure of 20-50 nm is introduced through the use of a mixed porogen in the suspension polymerization process, and a microporous structure of less than 20 nm is introduced into the cross-linking reaction after dangling double bonds, so that the adsorption While removing PBUTs, the agent also maintains a certain ability to remove medium-sized and small-molecule toxins. Moreover, the multi-component pore structure enables the adsorbent to maintain good adsorption performance while maintaining high mechanical strength and good adsorption kinetics. The introduction of capillary pores also improves the surface hydrophilicity and hydrophobicity and blood compatibility of the adsorbent. Compared with commercially available post-crosslinking macroporous adsorbents, the post-crosslinking reaction of pendant double bonds has simple steps, mild conditions, and reduces the use of a large amount of organic solvents, which is beneficial to environmental protection and cost reduction.

Claims (10)

1. An adsorbent for removing protein-bound uremic toxins by blood perfusion, characterized in that it is a porous resin with an amide group and with polystyrene-acrylonitrile-divinylbenzene as a skeleton, and the porous resin has The imprinted cavity of an imprinted molecule; the imprinted molecule includes protein-binding toxoids and/or analogs of protein-binding toxoids.
2. The adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 1, wherein the content of amide groups in the adsorbent is 1.0-2.5 mmol/g dry resin;

   Preferably, the particle size of the adsorbent is 0.4-1.2 mm;

   Preferably, the moisture content of the adsorbent is 50% to 70%;

   Preferably, the specific surface area of the adsorbent is 700-900 m2/g;

   Preferably, the sphericity of the adsorbent after grinding is greater than or equal to 90%;

   Preferably, the protein-bound toxoid comprises at least one of p-cresol sulfate and indoxyl sulfate;

   Preferably, the analog of the protein-bound toxoid includes at least one of p-toluenesulfonic acid and L-tryptophan.
3. The preparation method of the adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 1 or 2, characterized in that, comprising the following steps:

   (a) the oil phase is mixed with the water phase to carry out a polymerization reaction, and resin A is obtained after the separation;

   the oil phase includes styrene, acrylonitrile, divinylbenzene, porogen and benzoyl peroxide;

   the aqueous phase includes gelatin, sodium chloride and water;

   (b) in the resin A that step (a) obtains, add the vitriol oil to carry out hydrolysis reaction, obtain resin B;

   (c) mixing the ethanol solution of the imprinted molecule, the resin B, 1,2-dichloroethane and anhydrous ferric chloride to carry out a cross-linking-imprinting reaction to obtain a protein-bound uremic drug for blood perfusion removal Adsorbents for toxins.
4. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 3, wherein in step (a), the oil phase is mainly composed of the following components by mass : 5-15 parts of styrene, 5-15 parts of acrylonitrile, 70-90 parts of 80% divinylbenzene, 100-150 parts of porogen and 0.5-1.5 parts of benzoyl peroxide;

   Preferably, the aqueous phase comprises the following components in mass concentration: 0.5%-2% of gelatin and 5%-10% of sodium chloride;

   Preferably, the mass ratio of the water phase to the oil phase is (2.5-4):1;

   Preferably, the mass ratio of the imprinted molecules to the resin B is (1-4):20;

   Preferably, the mass ratio of the anhydrous ferric chloride and the resin B is (3-8):20;

   Preferably, the porogen includes component A and component B, the component A is selected from alkanes and/or aromatic hydrocarbons, and the component B is selected from alcohols and/or esters;

   Preferably, the component A is selected from at least one of toluene, ethylbenzene, xylene, n-heptane and 200# gasoline;

   Preferably, the component B is selected from at least one of cyclohexanol, isoamyl alcohol, n-octanol, dodecanol and butyl acetate;

   Preferably, the mass of the component A is 50% to 70% of the total mass of the porogen.
5. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 3, wherein in step (a), the initial mixing temperature of the oil phase and the water phase is 48～52℃;

   Preferably, the oil phase and the water phase are mixed and left to stand, and then stirred, heated and kept warm;

   Preferably, the standing time is 8-12 min;

   Preferably, the heating is carried out at a rate of 0.8-1.1°C/2min to 78-90°C;

   Preferably, the temperature is kept at 78-90° C. for 4-12 hours.
6. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 3, wherein in step (a), the separation comprises: performing solid-liquid separation on the reacted mixture , the obtained resin is washed with water, alcohol washed and sieved to obtain resin A;

   Preferably, the temperature of the water washing is 48-52°C.
7. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 3, wherein in step (b), the concentrated Sulfuric acid and stirring, after the solid-liquid separation of the reacted mixture, the separated resin is washed with gradient concentrated sulfuric acid, washed with water until neutral, and dried to obtain resin B.
8. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 7, wherein the time for adding the concentrated sulfuric acid at 20-25°C and stirring is 8-12 hours ;

   Preferably, the concentration of the concentrated sulfuric acid is 90% to 95%;

   Preferably, the drying temperature at which the resin B is obtained after drying is 70-78°C;

   Preferably, the resin B is dried to a moisture content of less than 2%.
9. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 3, wherein in step (c), the ethanol solution of imprinted molecules, the resin B, 1, The mixture of 2-dichloroethane is stirred and swollen, and then anhydrous ferric chloride is added and heated and heated and kept warm, and solid-liquid separation is performed to obtain an adsorbent for blood perfusion to remove protein-bound uremic toxins;

   Preferably, the temperature for stirring the mixture of the alcohol solution of the imprinted molecule, the resin B, and 1,2-dichloroethane is 28-32° C., and the time is 1.8-2.2 h;

   Preferably, the amount of the resin B is 20 parts by mass;

   Preferably, the alcohol solution of the imprinted molecules is composed of: 1-4 parts by mass of the imprinted molecules and 10-20 parts by volume of absolute ethanol;

   Preferably, the amount of the 1,2-dichloroethane is 80-120 parts by mass;

   Preferably, the heating temperature is increased to 65-80°C, and the holding time is 8-16h;

   Preferably, the amount of the anhydrous ferric chloride is 3-8 parts by mass.
10. The method for preparing an adsorbent for removing protein-bound uremic toxins by blood perfusion according to claim 9, wherein the resin after the solid-liquid separation described in the step (c) is washed with ethanol and water, and the The resin is packed into a column, washed with acetone-acid solution, washed with water until neutral, and dried;

    Preferably, the number of times of washing with ethanol is 2 to 3 times, and the number of times of washing with water is 2 to 3 times; the amount of each time is 180 to 220 parts by mass;

    Preferably, the acetone-acid solution is composed of acetone, water and hydrochloric acid in a volume ratio of (4.9-5.1):(3.9-4.1):1;

    Preferably, the dosage of the acetone-acid solution is 8-12 bed volumes.