JP5824873B2 - High mobility group protein adsorption carrier - Google Patents

Description

  The present invention relates to a high mobility group protein adsorption carrier.

  Hepatitis is a general term for diseases that cause inflammation in the liver and symptoms such as fever, jaundice, general malaise. Depending on the cause of the onset, viral hepatitis (cause of onset: A, B, C, D, Hepatitis E and G virus infection), alcoholic hepatitis (cause of alcohol consumption), drug-induced hepatitis (cause of development: drugs taken), autoimmune hepatitis (cause of development: abnormal immune system, especially autoantibodies, etc. Production)).

  In addition, hepatitis develops from the course of onset and symptoms, 1) acute hepatitis that suddenly develops and is a transient symptom, 2) liver function test abnormalities persist for more than 6 months, eventually cirrhosis and hepatocytes Chronic hepatitis that may progress to cancer 3) Special hepatitis among acute hepatitis, fulminant with hepatic encephalopathy within 8 weeks after first symptoms, and prothrombin time of 40% or less Hepatitis (serious hepatitis with a lifesaving rate of about 20% even after intensive treatment) is roughly classified.

  In the treatment of chronic hepatitis, the goal is to stop the transition to cirrhosis and cure at the stage of chronic hepatitis, so antiviral agents (interferon, lamivudine) that suppress hepatitis virus growth and liver protection that suppresses liver inflammation The administration is centered on drugs.

  In the treatment of acute hepatitis, it is fundamental to keep calm, and when there is no appetite, intravenous infusion of glucose center is given and nourishment is observed to wait for the symptoms to subside, but treatment of fulminant hepatitis However, blood purification therapy using plasma exchange that supplements coagulation factors to improve bleeding tendency and hemofiltration dialysis that supplements the liver's detoxification, etc., has been performed mainly by artificial liver adjuvant therapy.

  The main purpose of blood purification therapy for hepatitis treatment is to remove bilirubin, bile acids, creatinine and aromatic amino acids from the blood of hepatitis patients. For bilirubin, which is difficult to remove efficiently by hemofiltration dialysis, bilirubin Adsorbent and bilirubin removal methods have been devised and reported (Patent Documents 1 to 3).

  On the other hand, recently, it has been reported that the concentration of high mobility group protein is increased in the blood of patients and animal models such as acute hepatitis and fulminant hepatitis (Non-Patent Documents 1 to 4). High mobility group proteins are a group of non-histone DNA-binding proteins present in eukaryotic cells. HMGB-1, HMGB-2, HMGB-3, HMG-1L10, HMG-4L, SP100- HMG, HMGB-14, HMGB-17, and HMG-I (Y) are known as high mobility group proteins.

  Originally, the high mobility group protein was thought to be a factor that binds to DNA in the cell and is involved in the promotion of transcription and cell proliferation. However, AMPGB-1 is a factor that causes neurites to extend neurites. It has been reported that it has the property of stimulating monocytes and macrophages to induce inflammatory cytokines typified by interleukin-1β or interleukin-6. The possibility of having an action has been suggested (Non-Patent Documents 5, 6 and 7).

JP-A 63-275351 JP 02-286173 A Japanese Patent No. 3259860 JP 2007-29511 A JP 2005-296033 A International Publication No. 01/0742020

Zhang et al., J Huazhong Univ Sci Technology Med Sci., February 2008, 28, No. 1, p. 52-55 Takano et al., Japanese Society of Gastroenterological Surgery, July 2008, Vol. 41, No. 7, p. 1230 Liu et al., Zhonghua Gan Zang Bing Za Zhi., November 2007, Vol. 15, No. 11, p. 812-815 Takano et al., Shock. March 2010, p. 23 Jacek et al., Biochemistry, 2001, 40, 26, p. 7860-7867 Agello et al., Cytokine., May 2002, Vol. 21, No. 21, p. 231-236 Taniguchi et al., Arthritis Rheum. April 2003, Vol. 48, No. 4, p. 971-981

  However, the therapeutic effect of hepatitis by the existing blood purification therapy is not satisfactory, and even if the existing blood purification therapy is given to a patient with fulminant hepatitis, there are many cases where the patient dies. Furthermore, since treatment by plasma exchange involves the risk of viral infection, development of a safe treatment method with higher therapeutic effect is eagerly desired.

  On the other hand, although materials etc. which adsorb | suck a high mobility group protein are reported (patent documents 4-6), there is no report which suggests that adsorption removal of a high mobility group protein is effective in the treatment of hepatitis. There is no disclosure or suggestion of a carrier that can adsorb and remove inflammatory cytokines in addition to HMGB-1.

  Accordingly, the present invention provides a high mobility group protein adsorption carrier and a blood circulation column that can adsorb and remove inflammatory cytokines in addition to high mobility group protein in body fluids and can be used for treatment of hepatitis by blood purification therapy. It is aimed.

That is, this invention provides the high mobility group protein adsorption carrier and blood circulation column for hepatitis treatment as described in the following (1)-(7).
(1) A high mobility group protein adsorption carrier for treating hepatitis, wherein a functional group having an amino group is introduced on the surface of a water-insoluble carrier.
(2) The high mobility group protein adsorption carrier according to (1), wherein the functional group has an amide bond.
(3) The high mobility group protein adsorption carrier according to (1) or (2), wherein the functional group has a urea bond and / or a thiourea bond.
(4) The high mobility group protein adsorption carrier according to any one of (1) to (3), wherein the high mobility group protein is HMGB-1.
(5) one or more selected from the group consisting of interleukin-1β, interleukin-2, interleukin-6, interleukin-7, interleukin-8, monocytochemotactic protein 1 and macrophage inflammatory protein 1β The high mobility group protein adsorption carrier according to any one of the above (1) to (4), wherein the cytokine is adsorbed and removed.
(6) The high mobility group protein adsorption carrier according to any one of (1) to (5), wherein the shape is fiber, knitted fabric, felt, sponge, bead, or net.
(7) The high mobility group protein adsorption carrier according to any one of (1) to (6), wherein the hepatitis is acute hepatitis or fulminant hepatitis.
(8) A blood circulation column for treating hepatitis, which is packed with the high mobility group protein adsorption carrier according to any one of (1) to (7) above.

  According to the high mobility group protein adsorption carrier of the present invention, it is possible to adsorb and remove the high mobility group protein in the body fluid and inflammatory cytokines induced by the high mobility group protein at a high rate, and hepatitis due to blood purification therapy. Can be used to treat patients.

It is a figure which shows the survival curve of a pig hepatitis model. It is a figure which shows a time-dependent change of the HMGB-1 density | concentration in the blood sample of a porcine hepatitis model.

  The high mobility group protein-adsorbing carrier for treating hepatitis according to the present invention is characterized in that a functional group having an amino group is introduced on the surface of a water-insoluble carrier.

  “Hepatitis” refers to a pathological condition exhibiting inflammation in the liver, for example, drug-induced hepatitis, alcoholic hepatitis, hepatitis A, hepatitis B, hepatitis C, hepatitis D or hepatitis E, or sepsis (Sepsis derived from gram-negative bacteria, sepsis from gram-positive bacteria, culture-negative sepsis, fungal sepsis), influenza, acute respiratory distress syndrome (ARDS), acute lung injury (ALI) pancreatitis, inflammatory bowel disease (ulcerative colitis , Crohn's disease), transfusion of blood products, organ transplantation, reperfusion injury after organ transplantation, cholecystitis, cholangitis or hepatitis caused by neonatal blood group incompatibility, the above-mentioned high mobility group protein adsorption carrier, Among the above hepatitis, it is preferably used for the treatment of acute hepatitis or fulminant hepatitis.

  “High mobility group protein” (hereinafter “HMG protein”) is a non-histone DNA-binding protein present in body fluids, for example, HMGB-1, HMGB-2, HMGB-3, HMG-1L10, HMG-4L, SP100-HMG, HMGB-14, HMGB-17, or HMG-I (Y) may be mentioned, but for the purpose of treating hepatitis, it is preferable that HMGB-1 is removed from the body fluid. .

  In order to enhance the effect of treating hepatitis, it is preferable that the protein induced by HMG protein and the protein induced by inflammation are removed from the body fluid together with HMG protein.

  Examples of the protein induced by HMG protein and the protein induced by inflammation include TNFα, interleukin (hereinafter “IL”)-1α, IL-1β, IL-1Ra, IL-2, IL-6, IL. -7, IL-8, IL-10, IL-13, IL-18, IFN-γ, monocytochemotactic protein 1 (hereinafter “MCP-1”), macrophage inflammatory protein (hereinafter “MIP”) ) -1α, MIP-1β, MIP-2, MIF or PAF. For the purpose of treating hepatitis, IL-1β, IL-2, IL-6, IL-7, IL-8, It is preferable that MCP-1 and MIP-1β are removed from the body fluid.

  “Body fluid” refers to blood, plasma, serum, ascites or lymph, or a fraction component obtained from these or a liquid component derived from a living body.

  Examples of the “water-insoluble carrier” include polyolefins such as polyethylene (hereinafter “PE”) or polypropylene (hereinafter “PP”), polyesters such as polyethylene terephthalate or polybutylene terephthalate, and poly (p-phenylene ether sulfone). Polysulfone polymers, polyetherimides, polyimides, polyamides, polyethers, polyphenylene sulfides, polystyrene (hereinafter “PS”) or polyacrylonitrile polymers, or derivatives of these polymer compounds or blends of these polymers PS is preferable from the viewpoint of easy modification of the surface of the water-insoluble carrier by the amide methylation method, and PP or PP-PE copolymer is used from the viewpoint of heat resistance or maintaining the shape of the felt. Masui.

  The shape of the water-insoluble carrier is preferably fiber, knitted fabric, felt, sponge, bead or net.

  Among the above-mentioned water-insoluble carrier shapes, the knitted fabric, felt and net can be produced by a known method using fibers as raw materials. Examples of the felt production method include a wet method, a carding method, an air lay method, a spun bond method, and a melt blow method. Moreover, as a manufacturing method of a knitted fabric and a net, a plain weaving method or a tubular net method is mentioned, for example.

  Examples of the structure of the fiber include a single yarn or a core-sheath type, a sea-island type, or a side-by-side type composite fiber made of one type of polymer. A core sea-island type composite fiber or a sea-island type composite fiber in which the island is PP and the sea is PS is preferable. In order to impart strength and heat resistance to a water-insoluble carrier including fibers, it is also preferable to introduce a crosslinked structure with formaldehyde or paraformaldehyde or coat the surface with another polymer.

  When the shape of the water-insoluble carrier is a sheet-like felt or the like, the thickness is preferably 0.1 to 100 mm. When the shape is a knitted fabric or a net, the thickness is 0. 01-5 mm is preferable. If these water-insoluble carriers are wrapped around a central pipe and incorporated into a radial flow type module such as Tremixin (registered trademark; Toray Industries, Inc.), a felt is used to suppress the step difference between the winding start part and the winding end part. Is more preferably 10 mm or less, and more preferably 1 mm or less for knitted fabrics and nets.

  When the shape of the water-insoluble carrier is felt, it is preferable to take a laminated structure of two or more layers of a water-insoluble carrier and a net made of another material in order to improve shape retention.

  Examples of the laminated structure include a two-layer structure of a felt and a net, or a structure in which a net is sandwiched between felts, that is, a felt-net-felt sandwich structure. , “Body fluid etc.”) may be a multilayer structure as long as it does not affect the pressure loss when passing through these.

  Examples of the net for adopting the above laminated structure include a knot network, a knotless network, and a Raschel network. Moreover, as a shape of a mesh, a rectangle, a rhombus, or a turtle shell shape is mentioned, for example, As a magnitude | size, about 3 square mm is preferable.

  Examples of the net material for adopting the above laminated structure include polyamide, polyester, polyacrylonitrile-based polymer, PE or PP, and PP is particularly preferable in view of biocompatibility and steam sterilization resistance. For sterilization, polyester or PE is preferred.

  The net for adopting the above laminated structure is preferably formed of a monofilament so as to maintain strength and not increase pressure loss when body fluid or the like passes through the net.

  As for the thickness of the net | network for taking said laminated structure, 50-1200 micrometers is preferable.

  The diameter of the monofilament is preferably 50 to 1000 μm.

  Even when the shape of the water-insoluble carrier is a net, it can be wound around a central pipe to take a multilayer structure.

  Examples of the “amino group” include aminohexane, monomethylaminohexane, aminooctane, aminododecane, aminodiphenylmethane, 1- (3-aminopropyl) imidazole, 3-amino-1-propene, aminopyridine, aminobenzenesulfonic acid. , Tris (2-aminoethyl) amine, dimethylamine or diaminoethane, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, dipropylenetriamine, polyethyleneimine, N-methyl-2,2′-diaminodiethylamine, N-acetylethylenediamine An amino group derived from a compound having a plurality of amino groups such as 1,2-bis (2-aminoethoxyethane) (hereinafter referred to as “polyamine”) is preferable, and an amino group derived from polyamine is preferable. The amino group is preferably a primary or secondary amino group.

  The number of carbon atoms per nitrogen atom in the amino group is preferably 18 or less, more preferably 14 or less, and even more preferably 8 or less, considering nucleophilicity and steric hindrance affecting the reaction rate. The nitrogen atom of the amino group is preferably substituted with an alkyl group.

  If the amount of amino groups introduced into the water-insoluble carrier is too small, the desired adsorption performance cannot be obtained. On the other hand, if the amount is too large, the physical strength of the water-insoluble carrier is deteriorated and the adsorption performance tends to be lowered. Therefore, 0.03 μmol to 1 mmol per 1 g of dry weight of the water-insoluble carrier is preferable, and 0.1 μmol to 0.1 mmol is more preferable. In addition, when the water-insoluble carrier into which amino groups are introduced is PS, 0.1 μmol to 1 mol is preferable and 1 μmol to 500 mmol is more preferable per 1 mol of styrene which is a monomer constituting PS.

  A specific method for forming the “functional group having an amino group” includes, for example, a method in which tetraethylenepentamine or polyethyleneimine is reacted with PS having a chloroacetamidomethyl group as a reactive functional group. The polyethyleneimine used in the reaction is preferably a linear or branched polyethyleneimine having a molecular weight of 600 or more. However, since it is low in toxicity and easy to obtain and handle, a branched polyethyleneimine is more preferable. preferable.

  As the reactive functional group, in addition to a haloacetamidomethyl group such as chloroacetamidomethyl group, for example, an active halogen group such as a halomethyl group, a haloacetyl group or a halogenated alkyl group, an epoxide group, a carboxyl group, an isocyanate group , A thioisocyanic acid group or an acid anhydride group.

  The “functional group having an amino group” preferably further has an amide bond.

  As a method for further forming an amide bond in a functional group having an amino group, for example, a haloacetamidomethyl group is selected as the reactive functional group.

  The “functional group having an amino group” preferably further has a urea bond and / or a thiourea bond.

  Examples of a method for further forming a urea bond or a thiourea bond on a functional group having an amino group include a method of reacting a functional group having an amino group with isocyanate, isothiocyanate acid, acid chloride or acid anhydride. Is mentioned.

  The amount of isocyanate, isothiocyanate, acid, acid chloride or acid anhydride introduced in the above reaction is preferably 5 μmol to 300 mmol, more preferably 20 μmol to 100 mmol, per mol of the amino group to be reacted.

  Examples of the isocyanate or isothiocyanate include ethyl isocyanate, stearyl isocyanate, n-butyl isocyanate, iso-butyl isocyanate, n-propyl isocyanate, methyl isothiocyanate, ethyl isothiocyanate, n-butyl isothiocyanate, benzyl isothiocyanate, hexamethylene. Aliphatic isocyanate or aliphatic isothiocyanate such as diisocyanate, cyclohexyl isocyanate, cyclohexyl isothiocyanate or cyclohexyl diisocyanate or phenyl isocyanate, chlorophenyl isocyanate, fluorophenyl isocyanate, bromophenyl isocyanate, nitrophenyl isocyanate, tolyl isocyanate, methoxyphenol Nyl isocyanate, 1-naphthyl isocyanate, 4,4′-diphenylmethane diisocyanate, 3,3 ′, 5,5′-tetraethyl-4,4′-diisocyanatodiphenylmethane, phenyl isothiocyanate, chlorophenyl isothiocyanate, fluorophenyl isothiocyanate Aromatic isocyanate such as nitrophenyl isothiocyanate, tolyl isothiocyanate, methoxyphenyl isothiocyanate or 1-naphthyl isothiocyanate, or aromatic isothiocyanate, which controls useful nucleophilicity of amino groups and is useful in body fluids Aromatic isocyanate or isothiocyanate is preferable from the viewpoint of suppressing the adsorption of.

  As a specific method for further forming a urea bond to a functional group having an amino group, for example, a method in which tetraethylenepentamine and chlorophenyl isocyanate are reacted with PS having a chloroacetamidomethyl group as a reactive functional group. The amount of each reagent introduced in this method is preferably tetraethylenepentamine 3 μmol to 500 mmol, chlorophenyl isocyanate 5 μmol to 300 mmol, tetraethylenepentamine 1 μmol to 100 mmol, chlorophenyl isocyanate 20 μmol to 1 mol of styrene which is a monomer constituting PS. 100 mmol is more preferred.

  The HMG protein-adsorbing carrier for the treatment of hepatitis of the present invention is an HMG protein that is an adsorbed substance in a body fluid and a protein induced by the HMG protein (hereinafter referred to as “HMG protein etc.”) by treatment with the body fluid or the like for a certain time. Can be adsorbed.

The adsorption rate of the HMG protein and the like before and after the treatment in which the HMG protein-adsorbing carrier for treating hepatitis of the present invention is brought into contact with a body fluid for a certain time is calculated by the following formula 1.

Adsorption rate (%) = (C0−C) / C0 × 100 (1)
C0: Concentration of the adsorbate in the solution before treatment C: Concentration of the adsorbate in the solution after treatment

  The blood circulation column for the treatment of hepatitis of the present invention is characterized by being filled with the HMG protein adsorption carrier of the present invention.

  Examples of a method for treating body fluid using the blood circulation column of the present invention include, for example, connecting the blood circulation column of the present invention and a patient with a blood circuit, and allowing the body fluid taken out from the patient to pass through the blood circulation column of the present invention. An extracorporeal circulation method of returning this to the patient is preferable.

  The blood circulation column of the present invention may be used in combination with other body fluid treatment methods and medical devices. Examples of other body fluid treatment methods and medical devices include plasma exchange, peritoneal dialysis, plasma separator, hemofilter, heart-lung machine, and ECMO.

  The treatment time of the body fluid using the blood circulation column of the present invention is preferably a continuous treatment from the viewpoint of suppressing further inflammation induction by the HMG protein in the blood, more preferably 4 hours or more, and 24 hours or more. Further preferred.

  Hereinafter, the HMG protein adsorbing carrier and blood circulation column of the present invention will be specifically described with reference to experimental examples.

(Preparation of adsorption carrier A)
Sea-island type composite fiber (thickness: 2.6 denier, number of islands) consisting of 50 weight ratio sea component (mixture of 46 weight ratio PS and 4 weight ratio PP) and 50 weight ratio island component (PP) : 16; U.S. Pat. No. 4,661,260) was obtained from 50 g of N-methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 98% by weight sulfuric acid and 0.85 g of paraformaldehyde. It was immersed in the resulting mixed solution and reacted at 4 ° C. for 1 hour. The reaction fiber was immersed in 5 L of 0 ° C. ice water to stop the reaction, washed with water, and nitrobenzene adhering to the fiber was extracted and removed with methanol. The fiber was vacuum dried at 50 ° C. to obtain 71 g of a chloroacetamidomethylated crosslinked PS knitted fabric (hereinafter referred to as “AMPSt knitted fabric”).

Tetraethylenepentamine (1.5 g) was dissolved in dimethyl sulfoxide (hereinafter, “DMSO”) (500 mL), 20 g of AMPSt knitted fabric was added thereto with stirring, and the mixture was reacted at 25 ° C. for 6 hours. The AMPSt knitted fabric after the reaction was washed with 500 mL of DMSO on a glass filter. 3.0 g of AMPSt knitted fabric after washing is added to a solution of 150 mL of DMSO in which 1.0 g of parachlorophenyl isocyanate is dissolved, reacted at 25 ° C. for 1 hour, and then each of 60 mL of DMSO and distilled on a glass filter. It was washed with water and further washed with 3 L of distilled water and physiological saline, respectively, to obtain a knitted fabric (hereinafter referred to as “adsorption carrier A”) as an adsorption carrier. The structural formula of the functional group introduced into the adsorption carrier A is shown below.

(Preparation of adsorption carrier B)
Island core component: polypropylene, island sheath component: polystyrene 95% by mass, polypropylene 5% by mass, sea component: copolymer containing ethylene terephthalate unit as a main repeating unit and 3% by mass of 5-sodium sulfoisophthalic acid as a copolymerization component Polyester, composite ratio (mass ratio): core: sheath: sea = 45: 40: 15 After forming a felt composed of 85% by mass of fiber and 15% by mass of polypropylene fiber having a diameter of 20 μm, two pieces of this felt A sheet-like polypropylene net (thickness: 0.5 mm, single yarn diameter: 0.3 mm, opening: 2 mm square) was sandwiched between them, and needle punching was performed to form a three-layer structure.

Next, this three-layered felt is treated at 95 ° C. with a 3% by mass aqueous sodium hydroxide solution to make the ethylene terephthalate unit of the sea component as the main repeating unit, and 3% by mass of 5-sodium sulfoisophthalic acid as the copolymer component. By dissolving the copolyester contained, a three-layered felt having a core-sheath fiber diameter of 4.5 μm and a bulk density of 0.025 g / cm ³ was produced.

  50 g of the three-layer felt after the treatment with aqueous sodium hydroxide was immersed in a mixed solution consisting of 50 g of N-methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 98% by weight sulfuric acid and 0.85 g of paraformaldehyde, The reaction was carried out at 4 ° C. for 1 hour. The reaction fiber was immersed in 5 L of 0 ° C. ice water to stop the reaction, washed with water, and nitrobenzene adhering to the fiber was extracted and removed with methanol. This fiber was vacuum dried at 50 ° C. to obtain 71 g of a chloroacetamidomethylated crosslinked PS felt (hereinafter referred to as “AMPSt felt”).

Tetraethylenepentamine (5.0 g) was dissolved in 500 mL of DMSO, 20 g of AMPSt felt was added thereto with stirring, and the mixture was reacted at 25 ° C. for 6 hours. The AMPSt felt after the reaction is washed on a glass filter with 60 mL of DMSO and distilled water, respectively, and further washed with 3 L of distilled water and physiological saline, respectively. ) The structural formula of the functional group introduced into the adsorption carrier B is shown below.

(Preparation of adsorption carrier C)
11.4 g of polyethyleneimine (molecular weight 10,000; Wako Pure Chemical Industries, Ltd.) and 19 g of triethylamine were dissolved in 1000 g of DMSO, and 20 g of AMPSt knitted fabric was added thereto with stirring and reacted at 30 ° C. for 6 hours. The AMPSt knitted fabric after the reaction was washed with 60 mL of DMSO, methanol, and distilled water on a glass filter, respectively, to obtain a knitted fabric (hereinafter referred to as “adsorbing carrier C”) as an adsorbing carrier. The structural formula of the functional group introduced into the adsorption carrier C is shown below.

(Preparation of adsorption carrier D)
Cellulose fibers and N, N′-dimethylformaldehyde trioxide sulfate complex are mixed at 70 ° C. for 20 minutes, further mixed at 50 ° C. for 5 hours, neutralized with an alkaline solution, and finally washed with distilled water. As a result, a fiber in which sulfated ester groups were introduced at a density of 0.5% as the sulfur atom content (hereinafter referred to as “adsorption carrier D”) was obtained.

Example 1
In adult bovine serum, swine-derived HMGB-1 (initial concentration: 100 ng / mL) and Bio-Plex Human Cytokine Group I 17-Plex (BIO RAD) standard product (IL-1β (initial concentration: 1323 pg) / ML), IL-2 (initial concentration: 708 pg / mL), IL-6 (initial concentration: 1096 pg / mL), IL-7 (initial concentration: 1566 pg / mL), IL-8 (initial concentration: 841 pg / mL) ), MCP-1 (initial concentration: 865 pg / mL) and MIP-1β (initial concentration: 1134 pg / mL)) were prepared in an amount of 1 mL. 30 mg of adsorption carrier A, adsorption carrier B or adsorption carrier C was added thereto, and the mixture was treated at 37 ° C. for 2 hours using a rotator, and the adsorption rate before and after treatment was calculated for HMGB-1 and the like. The concentration of HMGB-1 was measured using HMGB1 ELISA Kit2 (Sinotest Co., Ltd.). In addition, the concentrations of IL-1β, IL-2, IL-6, IL-7, IL-8, MCP-1 and MIP-1β were measured by Bio-Plex Human Cytokine Group I 17-Plex (BIO RAD) Kit. And was measured using a Bio-Plex ™ workstation (BIO RAD). The results are shown in Table 1.

(Comparative Example 1)
By the same method as in Example 1, the adsorption rate before and after the treatment for HMGB-1 and the like was also calculated for the adsorption carrier D. The results are shown in Table 1.

  From the results in Table 1, one of the HMG proteins is the adsorption carriers B and C in which the functional group introduced on the surface of the water-insoluble carrier has an amino group, and the adsorption carrier A having a urea bond in addition to the amino group. Remarkable removal rate for any of HMGB-1 and IL-1β, IL-2, IL-6, IL-7, IL-8, MCP-1 and MIP-1β which are proteins derived from HMG protein It is clear that On the other hand, the adsorptive carrier D that has only the sulfated ester group as the functional group introduced on the surface of the water-insoluble carrier and has no amino group, urea bond or thiourea bond is constant for HMGB-1. However, other IL-1β, IL-2, IL-6, IL-7, IL-8, MCP-1 and MIP-1β all showed only a low removal rate.

(Preparation of blood circulation column A)
Adsorption carrier A was wound around a central pipe, and this was packed into a 165 mL capacity PP column case. After the inside of this column was washed with endotoxin-free water for 2 hours, a physiological saline solution was filled in the column, and finally 25 kGy of γ-ray sterilization was performed to prepare a blood circulation column A.

(Preparation of control column)
A PP column case with a capacity of 165 mL was filled with only physiological saline and sterilized with 25 kGy of γ-rays to prepare a control column.

(Example 2)
The blood circulation column A and the control column are washed with 1.5 L of physiological saline before use, and then primed with an aqueous solution obtained by adding 70 mg of the anticoagulant nafamostat mesylate to 0.5 L of physiological saline. I kept it.

  A hepatitis model was prepared by intravenously administering D-galactosamine at a capacity of 0.6 g / kg to male pigs (body weight 20-25 kg, N = 5), and using blood circulation column A and control column 20 hours after the administration. Then, extracorporeal blood treatment was performed under the condition of whole blood perfusion at a blood volume of 60 to 80 mL / min. In addition, heparin or nafamostat mesylate was added to blood as an anticoagulant, and the activated coagulation time (ACT) was maintained at about 150 to 200.

HMGB-1, IL-1β, IL-2, IL-6, IL-7, IL-8, MCP for blood samples collected at the beginning of the extracorporeal circulation process and blood samples at the end of the extracorporeal circulation process -1 and MIP-1β concentrations and ALT, AST, LDH, ALP and NH ₃ values were measured, respectively. For HMGB-1, a blood sample was collected every hour from the start of the extracorporeal blood treatment, and the concentration was measured. ALT, AST, LDH and ALP were measured by a method based on the JSCC standardization method, and NH ₃ was measured by a method based on the Okuda-Fujii modified method.

  The hepatitis pig after the extracorporeal circulation treatment was returned to the breeding cage to confirm the therapeutic effect after the extracorporeal circulation, and the survival situation was confirmed.

  Hepatitis pigs that have undergone extracorporeal blood treatment using blood circulation column A (hereinafter “pig A”), hepatitis pigs that have undergone extracorporeal blood treatment using control column (hereinafter “control pig”), For each, from the concentration of IL-1β, IL-2, IL-6, IL-7, IL-8, MCP-1 and MIP-1β in the blood sample at the start of extracorporeal circulation and at the end of extracorporeal circulation, The rate of change in the concentration of each protein was calculated. Specifically, the value obtained by dividing the concentration at the end of the extracorporeal circulation treatment by the concentration at the start of the extracorporeal circulation treatment was taken as the rate of change in the concentration of each protein. Table 2 shows the average value of the calculated change rate of each protein concentration.

  The survival curves prepared based on the respective death times of pig A and control pig are shown in FIG.

  From the results of Table 2 and FIG. 1, extracorporeal blood treatment was performed using blood circulation column A, and HMGB-1, IL-1β, IL-2, IL-6, IL-7, IL-8 were collected from the blood. It was revealed that the survival time of hepatitis pigs can be extended by removing both MCP-1 and MIP-1β at a high rate. That is, from the results of Table 2 and FIG. 1, it is clear that the HMG protein-adsorbing carrier of the present invention that can remove HMG protein and the like at a high rate has an excellent hepatitis treatment effect.

  FIG. 2 and Table 3 show the change rate of the HMGB-1 concentration in each blood sample of pig A and the control pig, that is, the change over time of the change rate of HMGB-1. The rate of change is the value obtained by dividing the HMGB-1 concentration in each sample by the HMGB-1 concentration at the start of extracorporeal circulation treatment, as in IL-1β, etc., and the average value is shown in FIG. 2 and Table 3. It was.

  From the results of FIG. 2 and Table 3, it is clear that the removal rate of HMGB-1 in the blood of pig A is significantly lower than that of the control pig.

For each of pig A and control pig, the rate of change of each value was calculated from the values of ALT, AST, LDH, ALP and NH ₃ of the blood sample at the start of the extracorporeal circulation treatment and at the end of the extracorporeal circulation treatment. Specifically, the value obtained by dividing the value at the end of the extracorporeal circulation process by the value at the start of the extracorporeal circulation process was defined as the rate of change of each value. Table 4 shows the average value of the calculated change rate of each value.

  From the results in Table 4, it is clear that the HMG protein-adsorbing carrier of the present invention that can remove HMG protein and the like at a high rate has an excellent hepatitis treatment effect.

  The present invention can be used as a blood circulation column for treating hepatitis in the medical field.

Claims (7)

A functional group having an amino group is introduced on the surface of the water-insoluble carrier ,
One or more cytokines selected from the group consisting of interleukin-1β, interleukin-2, interleukin-6, interleukin-7, interleukin-8, monocytochemotactic protein 1 and macrophage inflammatory protein 1β A high mobility group protein adsorption carrier for hepatitis treatment that is removed by adsorption.   The high mobility group protein adsorption carrier according to claim 1, wherein the functional group has an amide bond.   The high mobility group protein adsorption carrier according to claim 1 or 2, wherein the functional group has a urea bond and / or a thiourea bond.   The high mobility group protein adsorption carrier according to any one of claims 1 to 3, wherein the high mobility group protein is HMGB-1. The high mobility group protein adsorption carrier according to any one of claims 1 to 4 , wherein the shape is fiber, knitted fabric, felt, sponge, bead, or net. The high mobility group protein adsorption carrier according to any one of claims 1 to 5 , wherein the hepatitis is acute hepatitis or fulminant hepatitis. A blood circulation column for treating hepatitis, which is packed with the high mobility group protein adsorption carrier according to any one of claims 1 to 6 .

Images