CN110975842B

CN110975842B - Immunoadsorbent, preparation method thereof and adsorber for hemoperfusion

Info

Publication number: CN110975842B
Application number: CN201911294037.9A
Authority: CN
Inventors: 董凡; 房玉庆; 赵琪
Original assignee: Jafron Biomedical Co Ltd
Current assignee: Jafron Biomedical Co Ltd
Priority date: 2019-12-16
Filing date: 2019-12-16
Publication date: 2022-09-27
Anticipated expiration: 2039-12-16
Also published as: CN110975842A

Abstract

The invention provides an immunoadsorbent which comprises a carrier, a grafting connecting arm and a ligand, wherein the carrier is covalently connected with the grafting connecting arm, and the grafting connecting arm is connected with the ligand through a connecting group; the carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan, and the ligand is selected from tripterygium wilfordii extract and derivatives thereof. The invention also provides a preparation method of the immunoadsorbent and an adsorber for hemoperfusion by using the immunoadsorbent. Compared with the traditional administration mode, the triptolide provided by the invention can directly remove pathogenic factors in blood through blood circulation, can play a role in an extracorporeal blood perfusion device, cannot cause toxic damage to organ tissues of a patient in a mode of entering a human body, cannot generate drug resistance for the patient, and is convenient for medical care personnel to use.

Description

Immunoadsorbent, preparation method thereof and adsorber for hemoperfusion

Technical Field

The invention relates to the field of hemoperfusion adsorbents, in particular to an immunoadsorbent, a preparation method of the immunoadsorbent and an adsorber for hemoperfusion.

Background

The immunoadsorption is a conventional treatment means, which is to lead the blood of a patient out of the body and pass through a blood perfusion device, and eliminate pathogenic antibodies, antigens and immune complexes in the blood of the patient with autoimmune diseases by using an immunoadsorption agent in the blood perfusion device, so as to achieve the purposes of purifying the blood and relieving the disease conditions. The immunoadsorbent can remove redundant creatinine, beta 2 microglobulin, pathogenic antibodies, antigens, immune complexes and other substances in blood of patients with immune kidney diseases, can specifically remove Rheumatoid Factors (RF) in blood of patients with rheumatoid arthritis, reduces the deposition of the immune complexes in damaged joints and blood vessels, and eliminates or improves symptoms of fever, rash, arthralgia, arthroncus and the like of patients with rheumatoid arthritis.

Tripterygium wilfordii also known as Fibraurea recisa, belongs to the genus Tripterygium of Celastraceae. Clinical studies show that Triptolide (TP), also known as Triptolide and Triptolide, contained in tripterygium wilfordii is one of the most important immunosuppressive components contained in tripterygium wilfordii. Triptolide can inhibit interleukin-2 (IL-2) production and receptor effect, induce activated lymphocyte apoptosis but has no influence on thymocytes, interfere lymphocyte proliferation, inhibit activity of nuclear factor kappa B recombinant protein (NF-kappa B) and interleukin-2 (IL-2), block expansion of chronic inflammation of nephropathy, and relieve chronic inflammation change of renal tissue, thereby treating primary glomerulonephritis, purpuric nephritis, and lupus nephritis. Triptolide can also act on the largest subunit RPB1 in RNA polymerase II, inhibit gene transcription, combine with proteins such as human disintegrin-like metalloprotease-10 (ADAM10), deoxycytidine triphosphate pyrophosphatase-1 (DCTPP1), human group B xeroderma pigmentosum coupling factor recombinant protein (XPB) and the like, and down-regulate the molecular expression level of anti-apoptosis proteins such as anti-apoptosis protein Bcl-2, anti-apoptosis protein Bcl-xL, survivin Mcl-1, apoptosis inhibitor protein (IAP) family and the like, and has remarkable curative effect on rheumatoid arthritis.

However, triptolide has poor water solubility and high cytotoxicity, and clinical studies show that the effective dose is similar to the toxic dose, the drug tolerance of patients is not high, and the drug dose is difficult to control in clinical use. The triptolide injection is easy to cause phlebitis and even venous ulcer after entering a human body through intravenous injection. In addition, the oral preparation of triptolide has stimulation effect on gastrointestinal tract, and has great toxic effect on liver, kidney, heart, bone marrow, blood system, immune system, nervous system and the like of a patient. Because the quality of triptolide preparations on the current market is uneven, the curative effect of oral triptolide crude extracts is slow, and the blood concentration of patients cannot be effectively determined in real time, certain potential safety hazards exist in the use process of triptolide.

The Chinese patent ZL200410029615.3 discloses a hydrophilic polymer-Tripterygium wilfordii hook extract conjugate and a pharmaceutical composition thereof, wherein the conjugate improves the water solubility of the Tripterygium wilfordii hook extract or the Tripterygium wilfordii hook extract derivative, reduces the toxicity of the Tripterygium wilfordii hook extract or the Tripterygium wilfordii hook extract derivative, and prolongs the half life of the Tripterygium wilfordii hook extract in the circulation of an organism. Liu Chang et al (Liu Chang, Zhang Shi Rong. Synthesis of a conjugate of kidney-targeted triptolide and 2-glucosamine [ J ]. Huaxi, J.Pharmacology, 2017, Vol.32, No. 5, pp.452 to 455) disclose a conjugate of triptolide and 2-glucosamine with kidney targeting, and improve the water solubility of triptolide, can significantly reduce cytotoxicity and increase the rate of cellular uptake. The in vitro anti-tumor activity research on triptolide coupled by polyethyleneimine/cyclodextrin polymer [ J ]. Zhejiang university bulletin (medical edition), 2012, volume 41, stage 6, pages 24 to 33) discloses a triptolide-polyethyleneimine-cyclodextrin polycation compound, wherein the triptolide-polyethyleneimine-cyclodextrin polymer has reduced toxicity, has better capability of inhibiting cell transfer, and can effectively carry siRNA and enter cells. However, the three pharmaceutical compositions are still administrated by traditional modes such as oral administration, intranasal administration, rectal administration, transdermal administration or injection, and the like, and the stimulation and toxic effects of the drugs on human cells cannot be avoided; moreover, when the traditional mode is used for drug administration, patients are easy to generate drug resistance to the drugs, so that the drugs with normal dosage can not play the due effects any more, and even the drugs can completely lose efficacy, thereby bringing difficulty to the treatment of diseases.

The use of extracorporeal blood perfusion adsorption therapy has been widely used clinically. Triptolide is one of the first choice drugs for clinical treatment of rheumatoid arthritis, but no reports exist at present that triptolide is immobilized on an adsorbent to prepare an adsorbent and a blood perfusion device which can be used for adsorbing pathogenic substances of immune diseases such as rheumatoid arthritis, so that the adsorbent is necessarily developed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an immunoadsorbent, wherein the immunoadsorbent is filled in a column body to prepare an adsorber for hemoperfusion, and pathogenic antibodies, antigens and immune complexes in blood of patients with kidney diseases and rheumatoid arthritis are directly eliminated through blood circulation, so that the treatment effect is achieved.

The first aspect of the invention provides an immunoadsorbent, which comprises a carrier, a graft connecting arm and a ligand, wherein the carrier is covalently connected with the graft connecting arm, and the graft connecting arm is connected with the ligand through a connecting group. Specifically, the immunoadsorbent is obtained by reacting a carrier with a linker to obtain a graft carrier with a graft connecting arm, wherein the graft connecting arm is connected with a connecting group and then connected with a ligand through the connecting group. The carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan, and the ligand is selected from tripterygium wilfordii extract and derivatives thereof. According to the scheme, the tripterygium wilfordii extract and the tripterygium wilfordii derivative are grafted on the carrier to form the adsorbent, the adsorbent is loaded into an adsorber for extracorporeal blood purification to remove substances such as creatinine, beta 2 microglobulin, rheumatoid factors, immune complexes and the like in the blood of a patient, so that autoimmune diseases such as immune kidney diseases, rheumatoid arthritis and the like are treated, wherein the adsorbent has higher average adsorption rate on the rheumatoid factors RF-IgM, RF-IgG and RF-IgA, can obviously reduce the rheumatoid factors in the body of the rheumatoid arthritis patient, not only retains the biological activity of the tripterygium wilfordii extract and the tripterygium wilfordii derivative, but also avoids the direct toxic effect of the tripterygium wilfordii extract and the tripterygium wilfordii derivative on the human body, avoids the drug resistance of the patient to the adsorbent, and is very stable when the tripterygium wilfordii is bonded on the adsorbent carrier in a covalent bond way, the triptolide is not easy to fall off, so the risk of the triptolide entering the human body to generate toxic action is lower.

Preferably, the connecting group is one or more of an ester group, a carbonate group, an amide group, an ether group, an amine group, and an amino acid amide group. The chemical structures of the tripterygium wilfordii extract and the tripterygium wilfordii derivative all contain hydroxyl groups, and the tripterygium wilfordii extract and the tripterygium wilfordii derivative can be grafted on the surface of a carrier through one or more connecting groups such as ester groups, carbonate groups, amide groups, ether groups, amine groups and amino acid amide groups so as to achieve effective protection and reasonable utilization of ligand substances, the functional groups are all electronegative groups, and the remaining groups after ligand grafting can generate electrostatic adsorption force with rheumatoid factors to assist in strengthening the affinity of the adsorbent with the rheumatoid factors.

Preferably, the polystyrene-divinylbenzene is a porous macroporous resin. The porous macroporous resin can adsorb creatinine and beta 2 microglobulin in blood of a patient in a physical adsorption and chemical adsorption mode. The porous macroporous resin and the triptolide are matched, so that physical adsorption, chemical adsorption and biological affinity adsorption can be combined, and the adsorption performance of the adsorbent is improved.

Further preferably, the ligand is triptolide, and the connecting group is connected with the hydroxyl at position C14 on the triptolide. Triptolide is an epoxidized diterpene lactone compound with high activity in tripterygium wilfordii extracts, has various pharmacological effects such as anti-inflammation and immunosuppression, is one of main effective components for treating rheumatoid arthritis, is also lipophilic and hydrophobic, adsorbs rheumatoid factors through electrostatic binding and hydrophobic binding, and has a better adsorption effect. The C14 hydroxyl is a reaction site with stronger activity of triptolide, and the toxicity of the triptolide can be obviously reduced while the medicinal active structure of the triptolide is kept by esterifying and acetylating the C14 hydroxyl.

Still further preferably, the hydroxyl at C14 is attached to the linking group via an ester group. The ester group may be an ester group bonded to a hydrocarbon group, or an ester group moiety of an amide ester group or a carbonate ester group. The ester bonding mode is adopted to connect hydroxyl at C14 position of triptolide, so that the bioactivity of triptolide can be improved, and the adsorption performance is improved.

In a second aspect of the invention, there is provided a process for the preparation of an immunoadsorbent according to the first aspect of the invention, comprising the steps of:

step 1, carrying out a grafting reaction on the carrier and a connector under a catalytic condition to obtain a grafting carrier with a grafting connecting arm;

step 2, connecting the grafting carrier with the ligand to obtain the immunoadsorbent;

the carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan. The ligand is selected from radix Tripterygii Wilfordii extract and its derivatives.

In the preparation process of the adsorbent, a step of introducing a grafting connecting arm is firstly carried out, so that the adsorbent carrier is provided with the grafting connecting arm, and in the step of ligand grafting, the grafting connecting arm on the grafting carrier is connected with a ligand to form the adsorbent. The preparation process of the adsorbent does not involve chemical reaction with higher reaction requirement, so that the preparation process of the adsorbent is simpler and more efficient.

Preferably, the ratio of the carrier (volume/mL), the linker (mass/g) and the ligand (mass/g) is (1-5): (1-5): (0.002-0.005). The immunoadsorbent prepared by the ratio has good adsorption rate on rheumatoid factors RF-IgM, RF-IgG and RF-IgA.

Further preferably, step 1 specifically includes adding the carrier into a first solvent to swell for 3-5 hours, adding the linker and a first catalyst, carrying out sealed oscillation reaction at 0-37 ℃ for 12-24 hours, after the reaction is finished, washing with purified water for several times, and drying to obtain the grafting carrier with the grafting connecting arm, wherein the ratio of the carrier (volume/mL), the first solvent (volume/mL), the linker (mass/g) and the first catalyst (mass/g) is (1-5): (5-20): (1-5): (0.1-0.5).

The step 2 specifically comprises the following steps: dissolving the ligand into a second solvent, adding the grafting carrier and the second catalyst obtained in the step (1), carrying out sealed and light-proof oscillation reaction for 12-24 h at 0-25 ℃, and washing with alcohol and purified water for several times after the reaction is finished; wherein the ratio of the ligand (mass/g), the second solvent (volume/mL), the grafting carrier (volume/mL), and the second catalyst (mass/g) is (0.002-0.005): (5-20): (1-5): (0.001-0.002). The preparation process of the adsorbent is simple and efficient, and in the step of introducing the grafting connecting arm, the volume of the prepared grafting carrier is basically the same as that of the carrier.

Still further preferably, in step 1, the first solvent is one or more of DMF, dichloromethane, acetonitrile; the first catalyst is one or more of dimethylaminopyridine and pyridine. In step 2, the second solvent is one or more of DMF, dichloromethane and acetonitrile; the second catalyst is dimethylaminopyridine. In the step 2, a condensing agent and an acid-binding agent are also added into the reaction system, and the ratio of the condensing agent (mass/g), the catalyst (mass/g) and the acid-binding agent (mass/g) is (0.001-0.004): (0.001-0.002): (0.001-0.002). The raw materials used for preparing the adsorbent are easy to obtain, and the reaction rate is high.

Still further preferably, the carrier is polystyrene-divinylbenzene, and the preparation method further comprises a modification step, wherein the modification step is performed before the step 1, and the modification step is to perform chloromethylation on the polystyrene-divinylbenzene to obtain chloromethylated polystyrene-divinylbenzene; chloromethylated polystyrene-divinylbenzene was alcoholized. Polystyrene-divinylbenzene is lipophilic hydrophobic macroporous resin, triptolide is also lipophilic hydrophobic structure, and the polystyrene-divinylbenzene is used for adsorbing rheumatoid factors through electrostatic binding and hydrophobic binding and has good adsorption effect. The modified polystyrene-divinylbenzene has hydroxyl group for easy connection with linking group.

Still more preferably, the linker is one of acid anhydride, solid phosgene, amino acid, polyamine, and the acid anhydride is one or more of malonic anhydride, succinic anhydride, adipic anhydride, pyromellitic anhydride, and the like.

In a third aspect of the present invention, an adsorber for hemoperfusion is provided, wherein a column of the adsorber is filled with the immunoadsorbent according to the first aspect of the present invention or the immunoadsorbent prepared by the preparation method according to the second aspect of the present invention.

Compared with the traditional modes of oral administration, intranasal administration, rectal administration, transdermal administration or injection and the like, the triptolide can directly remove pathogenic factors in blood through blood circulation, can play a role in an extracorporeal blood perfusion device, cannot cause toxic damage to organ tissues of a patient in a mode of entering a human body, immediately relieves the illness state of the patient, cannot cause drug resistance of the patient, and is convenient for medical care personnel to use; can be used in combination with hemodialysis to reduce the pain of patients who take medicines every day, and can not generate the influence caused by forgetting to take medicines.

Drawings

FIG. 1 is a reaction scheme of chloromethylated polystyrene-divinylbenzene microsphere carrier in example 1 of the preparation method of immunoadsorbent of the present invention.

FIG. 2 is a reaction scheme of modified polystyrene-divinylbenzene in example 1 of the method for preparing an immunoadsorbent according to the present invention.

FIG. 3 is a reaction scheme showing the introduction of graft linker arms in example 1 of the method for preparing an immunoadsorbent of the present invention.

FIG. 4 is a reaction scheme showing the grafting ligand in example 1 of the method for preparing an immunoadsorbent of the present invention.

FIG. 5 is a schematic diagram of the configuration of the adsorber for hemoperfusion provided by the present invention, wherein 1 column and 2 immunoadsorbent are provided.

FIG. 6 is a reaction scheme showing the introduction of graft linker arms in example 2 of the method for preparing an immunoadsorbent of the present invention.

FIG. 7 is a reaction scheme showing the grafting ligand in example 2 of the method for preparing an immunoadsorbent of the present invention.

Fig. 8 is a structural diagram of the adsorbent in which triptolide is grafted with polystyrene-divinylbenzene with amino acid amide groups, obtained in example 9 of the preparation method of the immunoadsorbent of the present invention.

The invention is further explained with reference to the drawings and the embodiments.

Detailed Description

The invention provides an immunoadsorbent for blood perfusion, which is prepared by reacting a carrier with a linker to obtain a graft carrier with a graft connecting arm, wherein the graft connecting arm is provided with a connecting group and is connected with a ligand through the connecting group. The carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan, and the ligand is selected from tripterygium wilfordii extract and derivatives thereof.

The mechanism of action of the immunoadsorbent of the invention comprises pore size adsorption and chemisorption, of which chemisorption plays a major role. The carrier used in the invention is preferably a porous macroporous resin carrier, triptolide is of a lipophilic and hydrophobic structure, and creatinine and beta 2 microglobulin in blood of a patient are physically adsorbed in the adsorbent by the carrier through a physical adsorption mode of a molecular sieve and a chemical adsorption mode of hydrophobic binding, electrostatic binding and Van der Waals force with a specific aperture. The active pharmaceutical structure on the triptolide adsorbs rheumatoid factors through the combined action of physical adsorption, chemical adsorption and bioaffinity adsorption, including covalent bond, electrostatic binding, van der waals force, hydrophobic binding, space binding, complement binding, Fc segment binding and the like, so that the rheumatoid factors are adsorbed on the ligands.

The adsorbent, the carrier and the ligand of the invention act together to realize the specific adsorption of creatinine, beta 2 microglobulin and rheumatoid factor of nephropathy, thus solving the problems caused by the traditional administration of tripterygium wilfordii for treating rheumatoid arthritis in the prior art, such as stimulation and toxicity of the medicament to human cells; the patient is easy to generate drug resistance to the drug, so that the drug with normal dosage can not play the due role any more, even the drug can completely lose efficacy and the like. According to the invention, the ligand is grafted to the surface of the adsorbent carrier in a covalent bond manner, so that the triptolide is not easy to fall off, and the adsorbent is prepared into the blood perfusion device, so that the triptolide can play a role in the extracorporeal blood perfusion device, and toxic damage to organ tissues of a patient caused by a manner of entering a human body is avoided.

In addition, the adsorbent provided by the invention improves the adsorption rate of the hemoperfusion adsorbent to rheumatoid factors by grafting triptolide on the basis of adsorption of the adsorbent to creatinine and beta 2 microglobulin in the prior art, thereby realizing specific immunoadsorption.

The invention will be better understood by reference to the following detailed description of specific embodiments.

Example 1

The adsorbent carrier is polystyrene-divinylbenzene, the connector is pyromellitic anhydride, the ligand is triptolide, and the adsorbent carrier is grafted and immobilized with the triptolide based on an ester bonding mode, wherein the triptolide has a structural formula as follows:

1 adsorbent support preparation

1.1 preparation of chloromethylated polystyrene-divinylbenzene microspheres

The reaction route of chloromethylated polystyrene-divinylbenzene microsphere is shown in figure 1: suspending styrene and divinylbenzene for polymerization to obtain polystyrene-divinylbenzene; secondly, reacting the polystyrene-divinylbenzene with chloromethyl ether under the catalysis of zinc chloride to generate chloromethylated polystyrene-divinylbenzene.

The specific operation steps are as follows: styrene is used as a monomer, divinylbenzene is used as a cross-linking agent, polyvinyl alcohol (PVA) with the mass concentration of 0.5 to 3 percent is used as a dispersion medium, a pore-foaming agent is added, a reaction system is reacted at the temperature of 80 to 85 ℃, then toluene is removed by distillation, and then the polystyrene-divinylbenzene microsphere is obtained by continuously heating at the temperature of 95 to 100 ℃.

Adding 1 part by mass of the prepared polystyrene-divinylbenzene microspheres and 3-5 parts by mass of chloromethyl ether into a container; then standing at room temperature and adding anhydrous zinc chloride; then heating to 50-52 ℃ and reacting for 3-24 h; finally, cooling the reaction product to room temperature, filtering out mother liquor, washing the mother liquor for multiple times by using methanol, and drying the mother liquor to obtain the chloromethylated polystyrene-divinylbenzene microsphere.

1.2 Chloromethylated polystyrene-Divinylbenzol Alcoholation

The reaction route of the alcoholized polystyrene-divinylbenzene microspheres is shown in figure 2: the chloromethylated polystyrene-divinylbenzene microsphere is heated and hydrolyzed in sodium hydroxide water solution to prepare the alcoholized polystyrene-divinylbenzene microsphere. The specific operation steps are as follows: and (2) adding 10mL of chloromethylated polystyrene-divinylbenzene microsphere prepared in the step 1.1 into a 3mol/L sodium hydroxide aqueous solution, stirring and reacting for 24 hours at the temperature of 80-95 ℃, washing for multiple times by using purified water after the reaction is finished until the pH value of a washing solution is neutral, and drying to obtain the alcoholized polystyrene-divinylbenzene microsphere.

2 graft linker arm introduction

The reaction route of the polystyrene-divinylbenzene microsphere grafted with pyromellitic anhydride is shown in figure 3: carrying out grafting reaction on alcoholized polystyrene-divinylbenzene and pyromellitic anhydride under the catalysis of Dimethylaminopyridine (DMAP), wherein partial acid anhydride groups grafted on the carrier can be hydrolyzed into carboxylic acid groups in the reaction process. The specific operation process is as follows: adding 5mL of alcoholized polystyrene-divinylbenzene microsphere prepared in the step 1.2 into 20mL of N, N-Dimethylformamide (DMF) solvent for swelling for 2-3h, adding 5g of pyromellitic dianhydride and 0.3g of DMAP, carrying out sealed oscillation reaction at 37 ℃ for 24h, washing with purified water for several times after the reaction is finished, and drying to obtain the grafting carrier.

3 ligand grafting

The reaction scheme for the grafting ligands is shown in FIG. 4: the polystyrene-divinylbenzene microsphere grafted with pyromellitic anhydride and Triptolide (TP) are reacted under the catalysis of Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and triethylamine to prepare the polystyrene-divinylbenzene microsphere grafted with triptolide. The specific operation steps are as follows: dissolving 10mg triptolide in 20mL DMF solvent, adding 5mL grafted polystyrene-divinylbenzene microsphere prepared in the step 2, 2mg DCC, 1mg DMAP and 1mg triethylamine, sealing at room temperature, keeping out of the sun, shaking for reaction for 24h, and washing with alcohol and purified water alternately for several times after the reaction is finished to obtain the adsorbent.

4 adsorbent packing column

As shown in fig. 5, the adsorber for hemoperfusion comprises an adsorption column and an end cover, wherein two ends of the adsorption column are detachably connected with the end cover, a cavity 1 is arranged in the adsorption column, and the cavity is used for containing an immunoadsorbent 2 in the invention.

Example 2

The adsorbent carrier in example 2 is polystyrene-divinylbenzene microsphere, the linker is succinic anhydride, and the adsorbent carrier is grafted with triptolide based on ester group bonding mode. The polystyrene-divinylbenzene microsphere in example 2 also needs to be chloromethylated, which is the same as the chloromethylation method of the polystyrene-divinylbenzene microsphere in example 1.

1 adsorbent support preparation

1.1 modification of polystyrene-divinylbenzene support:

adding 20mL of chloromethylated polystyrene-divinylbenzene microsphere into a 4mol/L sodium hydroxide aqueous solution, stirring and reacting for 24h, washing for multiple times by using purified water after the reaction is finished until the pH value of a washing solution is neutral, and drying to obtain the alcoholized polystyrene-divinylbenzene microsphere.

2 graft linker arm introduction

The reaction route of polystyrene-divinylbenzene microsphere grafted with succinic anhydride is shown in figure 6: swelling alcoholized polystyrene-divinylbenzene microsphere in N, N-Dimethylformamide (DMF) solvent, adding succinic anhydride, and grafting succinic anhydride under the catalysis of 4-Dimethylaminopyridine (DMAP). The specific operation steps are as follows: adding 5mL of alcoholized polystyrene-divinylbenzene microsphere prepared in the step 1.1 into 20mL of DMF solvent for swelling, adding 5g of succinic anhydride and 0.3g of DMAP, carrying out sealed oscillation reaction at 37 ℃ for 24h, washing with purified water for several times after the reaction is finished, and drying to obtain the grafting carrier.

3 ligand grafting

The reaction scheme for the grafting ligands is shown in FIG. 7: the polystyrene-divinylbenzene microsphere grafted with the succinic anhydride and the Triptolide (TP) react under the catalysis of Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and triethylamine to prepare the polystyrene-divinylbenzene microsphere grafted with the triptolide. Dissolving 10mg of prepared triptolide in 20mL of DMF solvent, adding 5mL of polystyrene-divinylbenzene microspheres grafted with succinic anhydride, 2mg of DCC, 1mg of DMAP and 1mg of triethylamine prepared in the step 2, sealing at 25 ℃, avoiding light, performing oscillation reaction for 24 hours, and after the reaction is finished, alternately washing with alcohol and purified water for several times to obtain the adsorbent.

4 adsorbent packing column

The adsorbent prepared in the above 3 was loaded into an adsorber for hemoperfusion, and in this example, the adsorbent was installed in the adsorber in the same manner as in example 1.

Example 3

The main differences between this example 3 and example 2 are: the dosage of triptolide is 5mg, the preparation method of the adsorbent is the same as that in the example 2, and the arrangement mode of the adsorbent in the adsorber in the example is the same as that in the example 1.

Example 4

The main differences between this example 4 and example 2 are: the dosage of the succinic anhydride is 15g, the dosage of the triptolide is 25mg, the preparation method of the adsorbent is the same as that of the adsorbent in the example 2, and the arrangement mode of the adsorbent in the adsorber in the example is the same as that of the adsorbent in the example 1.

Example 5

In example 5, the adsorbent carrier is a combination of polystyrene-divinylbenzene and cellulose, the linker is succinic anhydride, the ligand is triptolide, and the carrier is grafted with triptolide based on an ester-based bonding manner, and since the cellulose contains a large amount of hydroxyl groups, the surface of the carrier does not need to be modified, and the cellulose can directly react with the linker and introduce the graft-linking arm.

1 graft linker arm introduction

Adding 5mL of the composition of polystyrene-divinylbenzene and cellulose into 20mL of N, N-Dimethylformamide (DMF) solvent for swelling, adding 5g of succinic anhydride and 0.3g of 4-Dimethylaminopyridine (DMAP), oscillating for 24h, washing with purified water for several times after the reaction is finished, and drying to obtain the grafting carrier.

2 ligand grafting

Dissolving 10mg of prepared triptolide in 20mL of DMF solvent, adding 5mL of the grafting carrier prepared in the step 1, 2mg of Dicyclohexylcarbodiimide (DCC), 1mg of 4-Dimethylaminopyridine (DMAP) and 1mg of triethylamine, sealing at room temperature and shaking for reaction for 24 hours in a dark place, and after the reaction is finished, alternately washing with alcohol and purified water for several times to obtain the adsorbent.

3 adsorbent packing column

The adsorbent prepared in 2 above was packed in an adsorber, and the adsorbent was arranged in the adsorber in the same manner as in example 1.

Example 6

The main differences between this example 6 and example 5 are: the carrier used was a combination of polystyrene-divinylbenzene and polyvinyl alcohol, used in an amount of 5mL, and the method of preparing the adsorbent was the same as that of example 5, and the adsorbent was disposed in the adsorber in the same manner as that of example 1.

Example 7

In example 7, the adsorbent carrier is a composition of polystyrene-divinylbenzene and polyvinyl alcohol, the linker is solid phosgene, the ligand is triptolide, and the carrier is grafted with triptolide based on a carbonate-based bonding manner, specifically:

1 introduction of graft linking arm

Dissolving 5mL of polystyrene-divinylbenzene and polyvinyl alcohol composition and 5g of solid phosgene in 10mL of anhydrous dichloromethane, dropwise adding 0.5mL of anhydrous pyridine, stirring at room temperature for 2 hours under the protection of nitrogen, rotationally evaporating to remove redundant solvent, washing with diethyl ether for 2-3 times, and drying in vacuum to obtain the grafting carrier.

2 ligand grafting

Adding 5mL of the obtained grafting carrier prepared in the step 1, 10mL of anhydrous dichloromethane, 10mg of triptolide, 1mg of 4-Dimethylaminopyridine (DMAP) and stirring for 8 hours at room temperature under the protection of nitrogen, filtering, removing redundant solvents by rotary evaporation, washing with diethyl ether for 2-3 times, and drying in vacuum to obtain the adsorbent, wherein the structure of the adsorbent obtained in the step 7 is as follows:

3 adsorbent packing column

The adsorbent prepared in 2 above was packed in a hemoperfusion cartridge, and the adsorbent was installed in the adsorber in the same manner as in example 1.

Example 8

In example 8, the adsorbent carrier is polystyrene-divinylbenzene, the linker is solid phosgene, the ligand is triptolide, and the carrier is grafted with triptolide based on an amide ester group bonding manner.

1 adsorbent support preparation

The polystyrene-divinylbenzene microsphere in this embodiment also needs to be modified, wherein the specific method for chloromethylating the polystyrene-divinylbenzene microsphere is the same as that for chloromethylating the polystyrene-divinylbenzene microsphere in embodiment 1.

1.1 amination of polystyrene-divinylbenzene microspheres

15mL of chloromethylated polystyrene-divinylbenzene microsphere and 40mL of ethylenediamine were added to the DMF solution and reacted for 7 hours. And (3) alternately washing the microspheres to be neutral by using ethanol and distilled water, and drying the microspheres to obtain the aminated styrene divinylbenzene microspheres.

2 graft linker arm introduction

Dissolving 15mL of aminated polystyrene-divinylbenzene microsphere prepared in the step 1.1 and 5g of solid phosgene in 60mL of anhydrous acetonitrile, dropwise adding 2mL of anhydrous pyridine, stirring for 2h under the protection of nitrogen, removing excess solvent by rotary evaporation, adding 50mL of diethyl ether to residual solid, filtering, and drying in vacuum to obtain the grafting carrier.

3 ligand grafting

Dissolving 5mL of modified polystyrene-divinylbenzene microsphere prepared in the step 2, 10mg of triptolide and 0.6g of DMAP in 60mL of anhydrous acetonitrile, adding 1mL of triethylamine, stirring at room temperature under the protection of nitrogen overnight, removing redundant solvent by rotary evaporation, filtering, and drying in vacuum to obtain the adsorbent of the polystyrene-divinylbenzene grafted triptolide with amide ester groups, wherein the structure of the adsorbent is shown in the figure:

4 adsorbent packing column

The adsorbent prepared in 3 above was loaded into an adsorber, and the adsorbent was installed in the hemoperfusion cartridge in this example in the same manner as the adsorbent in the adsorber of example 1.

Example 9

In example 9, the adsorbent carrier is polystyrene-divinylbenzene, the linker is glycine, the ligand is triptolide, and the carrier is grafted with triptolide based on amino acid amide bonding. The polystyrene-divinylbenzene microsphere in this example also needs to be modified, wherein the specific method for chloromethylation of polystyrene-divinylbenzene is the same as that for chloromethylation of the polystyrene-divinylbenzene microsphere in example 1;

1 adsorbent support preparation

1.1 alcoholization of polystyrene-divinylbenzene microspheres:

adding 12mL of chloromethylated polystyrene-divinylbenzene microsphere into 3mol/L sodium hydroxide aqueous solution, stirring and reacting for 24h, washing for multiple times by using purified water after the reaction is finished until the pH value of a washing solution is neutral, and drying to obtain the alcoholized polystyrene-divinylbenzene microsphere.

2 introduction of graft linking arm

Adding the alcoholized polystyrene-divinylbenzene microsphere prepared in the step 1.1 into 20mL of DMF solution dissolved with 0.1g of DCC and 0.1g of DMAP, adding 12g of glycine (amino protected by Fmoc), slowly controlling the dropping process, magnetically stirring, reacting at a low temperature for 3h, washing and drying to obtain the polystyrene-divinylbenzene microsphere grafted with amino acid, and removing the protecting group on the glycine.

3 ligand grafting

3.1 preparation of triptolide succinate

Dissolving 14mg triptolide and 28mg succinic anhydride in 0.5mL anhydrous pyridine, adding 1mg DMAP and 1mg triethylamine, reacting at room temperature for 24h, monitoring the reaction with BLC, and adding pure water and hydrochloric acid to adjust the pH to acidity after the reaction is finished. Extracting with dichloromethane, mixing organic phases, distilling under reduced pressure, and performing silica gel column chromatography to obtain triptolide succinate, which can be understood as tripterygium wilfordii derivative.

3.2 polystyrene-divinylbenzene grafted triptolide succinate of amino acids

Adding 12mL of amino acid grafted polystyrene-divinylbenzene prepared in the step 2 and 14mg of triptolide succinate monoester prepared in the step 3.1 into 15mL of anhydrous DMF, adding 0.1g of DCC and 0.1g of DMAP, stirring at room temperature for 24h, washing and drying to obtain the adsorbent of the polystyrene-divinylbenzene grafted triptolide with amino acid amide, wherein the adsorbent is specifically shown in FIG. 8.

4 adsorbent packing column

The adsorbent prepared in 3.2 above was loaded into an adsorber, and the adsorbent was installed in the adsorber in this example in the same manner as in example 1.

In examples 1 to 9, the linking group on the graft linker arm was linked to the hydroxyl group at position C14 on triptolide. The triptolide molecule contains 1 pentabasic unsaturated lactone ring and 3 epoxy groups, the groups can react under certain conditions, 12-13 epoxy groups are far away from the AB ring from the molecular model of the triptolide, the steric hindrance is smaller than that of the other two epoxy groups, and the triptolide is easy to be attacked by a nucleophilic reagent, but 12-13 epoxy groups are necessary groups of the triptolide with bioactivity, and C14 hydroxyl of the triptolide is a reaction site with stronger activity, and a large number of researches show that the toxicity of the triptolide can be obviously reduced while the medicinal active structure of the triptolide is kept by performing esterification and acetylation modification on the C14 hydroxyl of the triptolide.

The adsorbents obtained in the examples and the adsorbents obtained in the control experimental group were tested and compared for their adsorption performance, and 5mL of styrene divinylbenzene resin not grafted with triptolide was used in the blood perfusion device in the control experimental group to prepare the adsorbents.

The invention adopts an in-vitro static adsorption method to test the adsorption performance of the adsorbent on rheumatoid factors in serum of patients with rheumatoid arthritis, and the method specifically comprises the following steps:

first, 0.5mL of adsorbent is taken, and the mass ratio of the adsorbent: adding serum sample of rheumatoid arthritis patient at a ratio of 1:10(V/V, volume ratio), shaking for 2 hr, collecting upper layer serum sample after adsorption process is finished, and detecting. The detection method of the rheumatoid factors is enzyme linked immunosorbent assay (Elisa), a Roche full-automatic biochemical analyzer and a rheumatoid factor ELISA kit are used, and the operation method is carried out according to the kit instruction.

The specific results of the adsorption performance of the adsorbents in the examples and the adsorbents in the control experiment group are shown in table 1.

Table 1:

as can be seen from table 1, in examples 1 to 6, the adsorbent carriers are all connected with triptolide through ester groups, wherein the adsorbent carriers of examples 1 to 4 are polystyrene-divinylbenzene microspheres, the adsorbent carrier of example 5 is a composition of styrene-divinylbenzene and cellulose, and the adsorbent carrier of example 6 is a composition of styrene-divinylbenzene polyvinyl alcohol; as can be seen from the data in Table 1, the immunoadsorbents of examples 1 to 6 have high adsorption rates for the rheumatoid factors RF-IgM, RF-IgG and RF-IgA, and can significantly reduce the rheumatoid factors in the body of the rheumatoid arthritis patients. The adsorption performance of example 2 is better than that of examples 1 and 3, probably because the steric hindrance of the succinic anhydride connecting substance in example 2 is smaller than that of the pyromellitic anhydride in example 1, so that the rheumatoid factor can be conveniently contacted with the surface of the adsorbent; meanwhile, the dosage of the triptolide in the embodiment 2 is 10mg which is more than 5mg in the embodiment 3, the grafting amount is increased, so that the active sites on the surface of the adsorbent are increased, and the adsorption performance is better. The connector in example 4 is succinic anhydride and the ligand triptolide, but the adsorption performance is not outstanding, which may be that too many grafts on the surface of the adsorbent carrier can weaken the adsorption capacity of the carrier on the rheumatoid factor to a certain extent due to the larger steric hindrance of the succinic anhydride and the triptolide.

In addition, the adsorption performance of example 2 is also superior to that of examples 5 and 6, which is probably because polystyrene-divinylbenzene is a lipophilic and hydrophobic macroporous resin, and triptolide itself is also lipophilic and hydrophobic, and adsorbs rheumatoid factors by electrostatic binding and hydrophobic binding, while cellulose and polyvinyl alcohol in polystyrene-divinylbenzene and cellulose compositions and polystyrene-divinylbenzene and polyvinyl alcohol compositions are strong hydrophilic carriers and have slightly weaker adsorption performance to rheumatoid factors than structures in which both the carrier and the ligand are hydrophobic. Examples 7 to 9 are adsorbents prepared by respectively using three covalent bonds of carbonate group, amide group and amino acid amide group, and the adsorbents have good adsorption capacity for rheumatoid factors.

The comparison of the adsorption rate of the adsorbent in the control experiment group with the examples shows that the adsorbents in examples 1 to 9 have an improved average adsorption rate or an improved adsorption rate on a specific rheumatoid factor compared with the adsorbents in the control experiment group, so that the adsorption performance of the adsorbent on the rheumatoid factor can be further improved by grafting triptolide onto the adsorbent carrier, and the immunoadsorbent formed by grafting triptolide onto the adsorbent carrier can be applied to an adsorber for hemoperfusion, so that the adsorption performance can be ensured and the bioactivity of triptolide can be exerted.

The invention adopts static elution and high performance liquid chromatography to measure the grafting stability of triptolide in the adsorbent in each embodiment, and the specific test method is as follows: preparing 20mL of ethanol-water mixed solution with the density of (0.9373-0.9378) g/mL by using 95% ethanol and ultrapure water as eluent, circularly washing the adsorbent in each embodiment at the flow rate of 2mL/min, finishing washing after 1h, and calculating the content of triptolide in the eluent by using a high performance liquid chromatography test. Chromatographic conditions (chromatographic column: Elite Sinachrom ODSBP, 5 μm, 250 × 4.6mm, eluent: acetonitrile/water 30/70, 30min, flow rate 1.0mL/min, column temperature: 40 ℃; minimum detection limit concentration of triptolide is 0.1 μ g/mL); the specific results are shown in Table 2.

TABLE 2

Note: the content of triptolide in the eluent is lower than the lowest detection limit concentration of 0.1 mu g/mL

As shown in Table 2, the concentration of triptolide in the eluate obtained by performing the elution test on the adsorbent grafted with triptolide in each example is lower than 0.1 μ g/mL, so that the triptolide is considered to be bonded on the adsorbent carrier in a covalent bond manner, which is very stable and basically free from shedding, and thus the risk of the triptolide entering the human body to generate toxic effects is low.

Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, and it should be understood that various changes and modifications may be made by those skilled in the art, and any changes, equivalents, improvements and the like, which fall within the spirit and principle of the present invention, should be included in the scope of the present invention.

Claims

1. An immunoadsorbent, comprising: the carrier is covalently connected with the grafting connecting arm, and the grafting connecting arm is connected with the ligand through a connecting group; the carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan, the connecting group is one or more of an ester group, a carbonate group, an amide group, an ether group, an amine group and an amino acid amide group, the grafting connecting arm is one of anhydride, solid phosgene, an amino acid and polyamine, and the anhydride is one or more of malonic anhydride, succinic anhydride, adipic anhydride and pyromellitic anhydride; the ligand is selected from triptolide.

2. The immunoadsorbent of claim 1, wherein: the polystyrene-divinylbenzene is porous macroporous resin.

3. The immunoadsorbent of claim 1 or 2, wherein: the ligand is triptolide, and the connecting group is connected with a hydroxyl at the C14 position on the triptolide.

4. The immunoadsorbent of claim 3, wherein: the hydroxyl at the C14 position is attached to the linking group via an ester group.

5. A method for preparing the immunoadsorbent of any one of claims 1 to 4, comprising the steps of:

step 2, connecting the grafting carrier with the ligand to obtain the immunoadsorbent, wherein the ratio of the carrier (volume/mL), the connector (mass/g) and the ligand (mass/g) is (1-5): (1-5): (0.002-0.005);

the carrier is selected from polystyrene-divinylbenzene or a composition of the polystyrene-divinylbenzene and one or more of crosslinked polyvinyl alcohol, polypropylene, chitosan, cellulose, agaropectin and glucan; the ligand is selected from triptolide;

step 1 specifically includes adding the carrier into a first solvent for swelling for 3-5 h, adding the connector and a first catalyst, carrying out sealed oscillation reaction at 0-37 ℃ for 12-24 h, washing with purified water for several times after the reaction is finished, and drying to obtain the grafting carrier with a grafting connecting arm, wherein the ratio of the carrier (volume/mL), the first solvent (volume/mL), the connector (mass/g) and the first catalyst (mass/g) is (1-5): (5-20): (1-5): (0.1 to 0.5);

the step 2 specifically comprises the following steps: dissolving the ligand into a second solvent, adding the grafting carrier and a second catalyst obtained in the step 1, sealing at 0-25 ℃, avoiding light, performing oscillation reaction for 12-24 hours, and after the reaction is finished, alternately washing with alcohol and purified water for several times; wherein the ratio of the ligand (mass/g), the second solvent (volume/mL), the grafting carrier (volume/mL), and the second catalyst (mass/g) is (0.002-0.005): (5-20): (1-5): (0.001-0.002).

6. The method of claim 5, wherein:

in step 1, the first solvent is one or more of DMF, dichloromethane and acetonitrile; the first catalyst is one or more of dimethylaminopyridine and pyridine;

in step 2, the second solvent is one or more of DMF, dichloromethane and acetonitrile; the second catalyst is dimethylaminopyridine; a condensing agent and an acid-binding agent are also added into the reaction system, and the ratio of the condensing agent (mass/g), the catalyst (mass/g) and the acid-binding agent (mass/g) is (0.001-0.004): (0.001-0.002): (0.001-0.002).

7. The production method according to claim 5, characterized in that:

the carrier is polystyrene-divinylbenzene, the preparation method further comprises a modification step, the modification step is carried out before the step 1, and the modification step comprises the following steps:

chloromethylating polystyrene-divinylbenzene to obtain chloromethylated polystyrene-divinylbenzene;

chloromethylated polystyrene-divinylbenzene is alcoholized or aminated and the alcoholized polystyrene-divinylbenzene or aminated polystyrene-divinylbenzene is washed to neutral pH.

8. The production method according to any one of claims 5 to 7, characterized in that:

the connecting object is one or more of acid anhydride, solid phosgene, amino acid and polyamine, and the acid anhydride is one or more of malonic anhydride, succinic anhydride, adipic anhydride and pyromellitic anhydride.

9. An adsorber for hemoperfusion, comprising:

the adsorber contains an adsorbent according to any one of claims 1 to 4 or an adsorbent produced by the production method according to any one of claims 5 to 8.