JP2016077466A - Adsorbent for removing histone and device for purification of liquid derived from living organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a histone adsorbent and a device for purification of liquid derived from a living organism in which a histone in the liquid derived from the living organism can be removed efficiently.SOLUTION: An adsorbent is provided for removing a histone from a liquid derived from a living organism. The adsorbent is water insoluble and has a ligand connected to a water insoluble carrier, the ligand that has a hydrocarbon chain and/or a cyclic organic compound portion and the charge of 0 or more. Also, a device for purification of the liquid derived from the living organism is provided for removing the histone from the liquid derived from the living organism. The device for purification of the liquid derived from the living organism has a housing with an inlet and an outlet of the liquid derived from the living organism, and the adsorbent stored in the housing. The histone is removed out of the liquid derived from the living organism by the circulation of the liquid derived from the living organism in the housing.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an adsorbent that adsorbs histone in a biological fluid, and a biological fluid purification device using the same.

  Systemic Inflammatory Response Syndrome (SIRS) is defined as a state in which inflammation and other substances are triggered by excessive release of cytokines and other substances, and there is no effective treatment. There are 18 million people worldwide. Died every year.

  To date, clinical trials such as anti-TNF-α antibodies and IL-1Ra have been attempted for the purpose of neutralizing excessively produced inflammatory cytokines against diseases involving SIRS. It has not been proved yet.

  The reason why treatment attempts targeting single inflammatory cytokines have not shown effective results is because the background and symptoms of SIRS vary, and not only one type of cytokine causes SIRS, it is very complex This is thought to be due to the formation of a unique cytokine network.

  Blood purification therapy such as hemofiltration that comprehensively removes these pathogenic substances from the blood has attracted attention, and a technique for removing proteins including cytokines in the middle molecular region has been disclosed.

  In the past, blood purification therapy using filtration and dialysis has attempted to remove all mediators involved in complex cytokine networks, but it is also considered effective due to a mechanism different from the administration of cytokine monoclonal antibodies for multiple organ failure. However, there is a limit to the amount that can be removed.

  Therefore, in recent years, there has been a report on an inflammatory cytokine adsorbent for the purpose of treating SIRS by adsorbing and removing these inflammatory cytokines from the blood (see, for example, Patent Document 1). .

  Furthermore, recently, a study result that histone is very important as a pathogenic substance of SIRS has been disclosed (for example, see Non-Patent Documents 1 and 2).

  This can be verified from the fact that the blood histone concentration of SIRS patients is several tens to several hundred times higher than that of healthy individuals, and that the inflammation model mice recovered by administration of histone antibodies ( For example, refer nonpatent literature 3.).

  Histone is a major protein that constitutes eukaryotic chromatin and contains many basic proteins. Therefore, a long DNA molecule with a positive charge and a negatively-charged DNA strand is wrapped around twice. It has the role of folding and storing it in the nucleus. Histones are classified into four types, H2A, H2B, H3, and H4, and those in which DNA is wound around histone octamers that form two octamers each bonded together are called nucleosomes. On the other hand, histones that bind to DNA between nucleosomes (linker DNA) are called linker histones, and a typical one is called histone H1.

  These histones are known to be cationic. In fact, as disclosed in Patent Documents 2 and 3, one of separation and purification target proteins using an anionic adsorbent such as sulfated cellulose or hydroxyapatite. One.

Special table 2013-523772 JP2007-92034 JP-A-8-71412

Xu J1, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, Taylor FB, Esmon NL, Lupu F, Esmon CT, "Extracellular histones". 15, 1318-1321,1 Toshiaki Ichiba, Miwa Murai, Isao Nagaoka, Yoko Tabe, “Neurophil extracellular traps (NETs), damage-associated molecular patterns (DAMPs), and cell death in sepsis”, Journal of Japanese Emergency Medicine, 2013, 24, 836 Takashi Ito, Seiro Maruyama, Grants-in-Aid for Scientific Research (Academic Research Grants Grants) Research Report “Analysis of the Dynamism of a New Mediator of Sepsis“ Extracellular Histone ””

  Thus, histones originally have functions necessary for the living body. However, in pathological conditions such as sepsis, histones are substances that are released excessively outside the cells and cause pathological deterioration, resulting in death of the living body. However, histones are the main proteins that make up eukaryotic chromatin, and have a very important role in folding DNA molecules and storing them in the nucleus. However, administration of a drug that inhibits histone activity may cause serious side effects on the living body. That is, a means for selectively removing extracellular histones that are undesirable for a living body from the body is desired. Therefore, it is possible to remove histones by extracorporeal circulation.

  On the other hand, the adsorbents described in Patent Documents 2 and 3 have not been reported on examples in which the adsorption performance from biological fluids such as blood and plasma is actually measured while histone is used as a target substance. Then, when the present inventors investigated the histone adsorption ability of the adsorbents described in Patent Documents 2 and 3, the adsorbents described in Patent Documents 2 and 3 have low histone adsorptivity from the biological fluid, It was found to be inappropriate as a histone adsorbent.

  Therefore, the present inventors have conducted extensive studies to provide an adsorbent capable of efficiently adsorbing and removing histones from the biological fluid. As a result, the adsorbents described in Patent Documents 2 and 3 described above have adsorbability. As was not shown, in biological fluids, histones are not adsorbed on materials with only negative charges, which were previously thought to adsorb histones, and do not adsorb hydrocarbon chains and / or cyclic organic compound moieties. It has been found that histones can be efficiently adsorbed and removed by a material having a ligand having a ligand charge of 0 or more.

The present invention is a histone adsorbent and a biological fluid purification device shown in the following (1) to (8).
(1) An adsorbent that removes histone from a biological fluid, wherein a ligand is immobilized on a water-insoluble carrier, the ligand having a hydrocarbon chain and / or a cyclic organic compound moiety, and An adsorbent in which the charge of the ligand is 0 or more.
(2) The adsorbent according to (1), wherein the charge of the ligand is 0.
(3) The adsorbent according to (1) or (2), wherein the ligand has a negative charge with a hydrophobic atomic group.
(4) The adsorbent according to any one of (1) to (3), wherein the ligand is an amino acid.
(5) The adsorbent according to any one of (1) to (4), wherein the amino acid is tryptophan or phenylalanine.
(6) The adsorbent according to any one of (1) to (5), wherein the water-insoluble carrier of the adsorbent is a porous bead carrier made of polyvinyl alcohol gel.
(7) The adsorbent according to any one of (1) to (6), wherein the histone is histone H3.
(8) A biological fluid purification device for removing histone from biological fluid, a housing provided with an inlet and an outlet for biological fluid, and (1) to (7) accommodated in the housing A biological fluid purifying device, comprising: the adsorbent according to any one of the above, and removing histone from the biological fluid by circulating the biological fluid.

  According to the present invention, a histone adsorbent and a biological fluid purification device are provided.

It is sectional drawing which shows one Embodiment of the biological body origin liquid purification device. It is sectional drawing which shows another embodiment of the biological fluid purification device.

  Hereinafter, preferred embodiments of the present invention (hereinafter referred to as “present embodiments”) will be described in detail. In addition, this embodiment shown below illustrates the apparatus and method for embodying the technical idea of this invention, and the technical idea of this invention specifies the combination etc. of a structural member to the following. Not what you want.

  The adsorbent according to the present embodiment is an adsorbent that removes histone from a biological fluid, wherein a ligand is immobilized on a water-insoluble carrier, and the ligand includes a hydrocarbon chain and / or a cyclic organic compound moiety. And an adsorbent in which the charge of the ligand is 0 or more. Further, the biological fluid purification device according to the present embodiment is a biological fluid purification device for removing histones from biological fluid, and includes a housing having an inlet and an outlet for biological fluid, and a housing. An adsorbent containing a ligand immobilized on a water-insoluble carrier, wherein the ligand has a hydrocarbon chain and / or a cyclic organic compound moiety, and the charge of the ligand is 0 or more; And the biological fluid is circulated in the housing to remove histone from the biological fluid.

(Carrier)
The adsorbent according to this embodiment includes a water-insoluble carrier. By being insoluble in water, it is possible to prevent the carrier material from being eluted into the blood when the adsorbent comes into contact with blood. In this specification, “water-insoluble” means a weight loss after the adsorbent in a dried state dried for 24 hours is immersed in pure water for 24 hours and then taken out and dried at 60 ° C. for 24 hours. It means 10.0 wt% or less.

  The shape of the adsorbent is not limited, and may be any of particulate, hollow fiber, nonwoven, fiber, sheet, and sponge, but may be particulate from the viewpoint of contact surface area with the biological fluid. desirable.

  In consideration of the specific surface area and the flowability of the biological fluid, the particulate carrier preferably has an average particle size of 50 to 1500 μm, and more preferably 100 to 1000 μm for the purpose of allowing whole blood to pass through. . Here, the particle diameter means the length of the longest straight line among straight lines connecting any two points on the outline of the carrier when observed with a microscope. The average particle diameter means an arithmetic average value of the particle diameters of 100 particulate carriers selected at random.

From the viewpoint of ensuring sufficient histone adsorption performance, the surface area of the particulate carrier is, for example, 1 m ² / g or more, preferably 5 m ² / g or more. As described later, the surface area of the carrier can be measured after removing the ligand from the adsorbent.

The surface area of the carrier is the surface area calculated using a BET (JIS Z 8830; specific surface area measurement method for powder (solid) by gas adsorption) analysis method after measuring the nitrogen adsorption isotherm at liquid nitrogen temperature (77K). Point to. There are two methods for measuring the nitrogen adsorption isotherm: a volumetric method for calculating the amount of nitrogen adsorbed on the carrier due to a pressure drop of the nitrogen gas filled in the cell, and a gravimetric method for measuring the weight change of the carrier. It is common that there is not much difference regardless of which measurement is used. If there is a difference in the measured surface area depending on the measurement method, it can be said that if any measured value is 1 m ² / g or more, sufficient histone adsorption performance can be secured. The same applies to the case where it can be evaluated that the surface area is 1 m ² / g or more by any method capable of measuring the surface area.

  When using a hollow fiber-shaped carrier, the inner diameter of the hollow fiber is preferably 100 to 300 μm from the viewpoint of flowing blood inside the hollow fiber.

  When the carrier is a nonwoven fabric or a woven fabric, the average fiber diameter can be 0.3 μm to 10 μm, preferably 0.3 μm to 3 μm, and more preferably 0.5 μm to 1.8 μm. . When the average fiber diameter is 0.3 μm or more, the pressure loss when the blood is filtered tends to be moderate, and red blood cells tend not to be hemolyzed.

  Here, the average fiber diameter is an average diameter measured from a photograph obtained by sampling a part of the nonwoven fabric or woven fabric constituting the filter carrier and observing with an electron microscope.

  Examples of carrier materials include cellulosic gels, dextran gels, agarose gels, polyacrylamide gels, porous glass, vinyl polymer gels, etc., which can be used as long as they are insoluble in water. From the viewpoint of ease of introduction, polyvinyl alcohol gel is preferably used.

  Examples of these carriers include cellulose such as Asahi Kasei Microcarrier (manufactured by Asahi Kasei Co., Ltd.), CM-Cellulofine (registered trademark) CH (exclusion limit protein molecular weight: about 3 × 10 6, sold by Seikagaku Corporation). -Based carriers, polyvinyl alcohol-based carriers such as a total porous activated gel described in JP-B-1-44725, CM-Toyopearl (registered trademark) 650C (exclusion limit protein molecular weight: 5 × 106, manufactured by Tosoh Corporation), etc. , CM-Trisacryl M (CM-Trisacryl M, exclusion limit protein molecular weight: 1 × 10 7, Pharmacia-LKB, Sweden) and other polyacrylamide carriers, Sepharose CL-4B (Sepharose CL-4B, Exclusion limit protein molecular weight: 2 × 107, Sweden Organic carrier such as agarose carrier such as Pharmacia-LKB (Nippon Pharmacia-LKB), and CPG-10-1000 (exclusion limit protein molecular weight: 1 × 10 8, average pore size: 100 nm, US electro-nucleo Examples thereof include inorganic carriers such as porous glass such as Nix (Electro-nucleonics).

  The composition of the carrier may be a known polymer that can take a porous structure, such as a polymer of polyamide, polyester, polyurethane, polysulfone, polyacrylonitrile, polymethyl methacrylate, and vinyl compound. . Alternatively, the adsorbent may be formed by immobilizing a ligand on a polymer-coated carrier made of these polymers.

(Immobilization of ligand)
In this specification, “ligand” refers to a substance or atomic group that exhibits an interaction (binding property) with a specific substance, and the structure is particularly limited as long as it has an affinity for the specific substance. There is no. In the present specification, a substance having an interaction with histone is particularly called a ligand.

  Whether or not the ligand has an interaction with the histone is determined by, for example, immersing a carrier on which the ligand is immobilized in a histone solution having a known concentration, and then measuring the histone concentration in the histone solution. It can be determined from the concentration change before and after immersion. If the histone concentration of the histone solution decreases by 10% or more before and after the immersion, this substance interacts with the histone.

  The method for immobilizing the ligand on the carrier is not particularly limited, but a method of forming a covalent bond is preferred. For example, when the carrier has a carboxyl group, it is converted to a succinimideoxycarbonyl group by reacting with N-hydroxysuccinimide, and when the ligand has an amino group, the ligand is A method of reacting with an amino group moiety (active ester method) can be mentioned. When the carrier has an amino group or carboxyl group, in the presence of a condensation reagent such as dicyclohexylcarbodiimide, when the ligand has an amino group or carboxyl group, the ligand carboxyl group or amino group is condensed. And a method of binding a carrier and a ligand using a compound having two or more functional groups such as glutaraldehyde. In addition, when the carrier has a hydroxyl group, a cyanogen halide such as cyanogen bromide is allowed to act on the carrier, and a method of reacting with the amino group portion of the ligand or an epoxide such as epichlorohydrin is allowed to act on the carrier, Examples thereof include a method of reacting with the amino group part or hydroxyl part of the ligand. For example, when the ligand is tryptophan, tryptophan can be immobilized on the carrier by reacting an epoxide such as epichlorohydrin with the carrier and reacting the amino group of tryptophan with the epoxy group of the porous body.

  The ligand can also be immobilized by physically coating the carrier with the ligand. For example, a method in which a carrier is immersed in a solution in which a ligand is dissolved in an appropriate solvent (ligand solution) for a certain period of time, or a method in which a ligand solution is sprayed onto the carrier can be used.

  The amount of ligand immobilization is determined by measuring the absorbance spectrum at a wavelength of 200 nm to 280 nm of a ligand solution in which the ligand used for the ligand immobilization reaction is dissolved, and using the wavelength showing the highest absorbance before and after the ligand immobilization reaction. The amount of immobilization per 1 mL gel of carrier (eq / mL gel) is calculated from the difference in absorbance of the ligand reaction solution. For example, when the ligand is tryptophan, the absorbance at 278 nm of the tryptophan solution to be used for the ligand immobilization reaction is measured, and the amount of immobilization per 1 mL gel (eq) is determined from the difference in the absorbance of the tryptophan solution before and after the ligand immobilization reaction. / ML gel).

  Alternatively, the amount of immobilization per 1 mL gel (mg / mL) may be calculated from the difference in the weight of the ligand solution before and after the ligand immobilization reaction of the ligand solution.

  In addition, after measuring the absorbance spectrum of a solution of an appropriate reagent such as sodium hydroxide at a wavelength of 200 nm to 280 nm, the ligand is eluted into this solution by hydrolysis reaction or the like, and the obtained ligand eluate having a wavelength of 200 nm to 280 nm is used. Measure the absorbance spectrum. Using the wavelength exhibiting the highest absorbance, the amount of immobilization per 1 mL gel (eq / mL gel) is calculated from the difference in absorbance of the ligand eluate before and after elution of the ligand. Alternatively, the amount of immobilization per 1 mL gel (mg / mL) may be calculated from the difference in the weight of the ligand eluate before and after elution of the ligand.

  With respect to the structure analysis of the ligand, it is possible to analyze the structure using various methods such as HPLC, elemental analysis, and NMR using the ligand eluate as a sample.

  In immobilizing the ligand, a molecule (spacer) having an arbitrary length can be introduced between the water-insoluble carrier and the ligand as necessary. Examples of the spacer molecule include a polymethylene chain and a polyethylene glycol chain. For example, the hydroxyl group of agarose and the isocyanate group on one side of hexamethylene diisocyanate can be reacted and bonded, and the remaining isocyanate group and the amino group, hydroxyl group, carboxyl group, etc. of the ligand can be reacted and bonded.

  The amount of ligand bound to the carrier, that is, the amount of ligand retained (the amount of immobilized ligand) is preferably in the range of 10 μmol to 200 μmol, more preferably in the range of 10 μmol to 100 μmol, per 1 mL gel of the carrier. . When the retention amount is 10 μmol or more, since the ligand can cover the entire support surface, the area where plasma is in direct contact with the support surface is reduced, nonspecific adsorption of useful plasma components is less likely to occur, and histones are more selectively selected. This is preferable because it tends to be adsorbed. In addition, when the amount is 200 μmol or less, ligands on the surface of the carrier are not excessively concentrated, and aggregation of the ligand can be suppressed. Therefore, nonspecific adsorption of useful plasma components hardly occurs, and histone tends to be more selectively adsorbed. Therefore, it is preferable.

(Hydrocarbon chain)
The hydrocarbon chain described in this specification refers to a molecular partial structure composed of carbon atoms and hydrogen atoms.
For example, when a carbon atom at the end of a molecular partial structure consisting of carbon atoms and hydrogen atoms is not covalently bonded to an atom other than a hydrogen atom, for example, a molecular partial structure consisting of one carbon atom and hydrogen is usually a methyl group. If the carbon atom at the end of the molecular substructure consisting of a carbon atom and a hydrogen atom is covalently bonded to a new atom other than a hydrogen atom, it is composed of one carbon atom and hydrogen. The molecular chain is a hydrocarbon chain usually called a methylene chain.

  The hydrocarbon chain described in the present specification may be a saturated hydrocarbon chain or an unsaturated hydrocarbon chain, and a saturated hydrocarbon chain and an unsaturated hydrocarbon chain may be mixed.

1) In the case of a saturated hydrocarbon chain When a carbon atom and a carbon atom are not connected by a double bond or a triple bond, and an acyclic saturated hydrocarbon chain is at the end of the molecule,
-(CnH2n + 1) where n is a group that can be represented by an integer of 1 or more, for example, a methyl group when n = 1 and an ethyl group when n = 2.
Also, if the acyclic saturated hydrocarbon chain is in the middle of the molecule (the carbons at both ends of the hydrocarbon chain are covalently bonded to atoms other than hydrogen)
— (CnH2n) — However, n is a molecular chain that can be represented by an integer of 1 or more. The acyclic saturated hydrocarbon chain may be linear or branched.
If a cyclic saturated hydrocarbon of 3 or more carbon atoms is at the end of the molecule,
-(CnH2n-1) where n is a group that can be represented by an integer of 3 or more.
If it is in the middle of the molecule,
-(CnH2n-2)-However, n is a molecular chain that can be represented by an integer of 3 or more.

2) In the case of an unsaturated hydrocarbon chain In the following, the formula in the case of having only one double bond or triple bond is described as an example, but the hydrocarbon chain has two double bonds or triple bonds. It also includes more than two cases.
If the acyclic unsaturated hydrocarbon chain with carbon-carbon double bond is at the end of the molecule,
-(CnH2n-1) where n is a group that can be represented by an integer of 2 or more.
If the acyclic unsaturated hydrocarbon chain is in the middle of the molecule,
-(CnH2n-2)-However, n is a molecular chain that can be represented by an integer of 2 or more.
When it has a carbon-carbon triple bond and an acyclic unsaturated hydrocarbon chain at the end of the molecule,
-(CnH2n-3) where n is a group that can be represented by an integer of 2 or more.
If it is in the middle of the molecule,
-(CnH2n-4)-However, n is a molecular chain that can be represented by an integer of 2 or more.
If the cyclic unsaturated hydrocarbon chain with a double bond is at the end of the molecule,
-(CnH2n-3) where n is a group that can be represented by an integer of 3 or more.
If it is in the middle of the molecule,
-(CnH2n-4)-However, n is a molecular chain that can be represented by an integer of 3 or more.
If a cyclic unsaturated hydrocarbon chain with a triple bond is at the end in the molecule,
-(CnH2n-5) where n is a group that can be represented by an integer of 3 or more.
If it is in the middle of the molecule,
— (CnH2n-6) — However, n is a molecular chain that can be represented by an integer of 3 or more.

  The hydrocarbon chain also includes alkyl groups, alkenyl groups, and alkynyl groups. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, and an icosyl group. Any group can be used. Further, it may be linear or branched. These functional groups can be employed singly or in combination.

  Examples of alkenyl groups include ethenyl group, propenyl group, butenyl group, pentenyl group, hexenyl group, heptenyl group, octenyl group, nonenyl group, decenyl group, icosenyl group, etc., and any alkenyl group can be used. . Further, it may be linear or branched. These functional groups can be employed singly or in combination.

  Examples of the alkynyl group include an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a nonynyl group, and a decynyl group, and any alkynyl group can be used. Further, it may be linear or branched. These functional groups can be employed singly or in combination.

(Cyclic organic compound part)
Some of the above-mentioned carbon hydrogen chains are included, but the cyclic organic compound part includes cyclohexane, benzene, naphthalene, adamantane, lactone, pyridine, cyclopentane, thiophene, thienylalanine, thienylglycine, thienylserine, porphyrin Indole ring, tryptamine, tocophenols represented by vitamin E, and the like, but any organic compound having a cyclic structure based on a carbon skeleton can be used, and includes condensed rings, heterocyclic rings, and the like. The ligand may have a cyclic alkyl group or a (hetero) aryl group alone or in combination, and may further have an alkyl group.

(Charge with ligand (E))
The charge (E) per molecule of the ligand is 0 or more. The charge (E) here is
Formula: E = (number of functional groups having a positive charge in the ligand) − (number of functional groups having a negative charge in the ligand)
Defined by Further, when the charge of the ligand is increased, that is, when the positive charge is excessive, the adhesion of blood cells and blood coagulation tend to occur. Therefore, the charge of the ligand is preferably 0. The charge (E) per molecule of the ligand is determined from the chemical formula of the ligand.

(Positive charge)
In the present specification, “having a positive charge” refers to being positively charged by being ionized in a neutral (pH 7.0) aqueous solution. For example, functional groups having a positive charge include functional groups such as amino groups, guanidyl groups, and amidine groups.

(Negative charge)
In the present specification, “having a negative charge” refers to being negatively charged by ionization in a neutral (pH 7.0) aqueous solution. For example, functional groups having a negative charge include functional groups such as carboxyl groups and sulfone groups.

  For example, when the ligand is phenylalanine, since the molecule contains an amino group and a carboxy group, the charge (E) of the ligand is E = 1-1 = 0.

(Ligand with negative charge)
From the viewpoint of suppressing fibrinogen adsorption, the ligand preferably has a negative charge. In this specification, “having a negative charge” means having a functional group having the negative charge described above.
As described above, since the charge (E) of the ligand is 0 or more, in order for the ligand to have a negative charge, the ligand needs to include a positive charge in excess of the number of negative charges. A positive charge and a negative charge may be contained in one hydrocarbon chain and / or cyclic organic compound part, or may be contained in a plurality of types of hydrocarbon chains and / or cyclic organic compound parts, respectively. Good.
If it is a ligand which has either of the functional groups which have a negative charge, there exists an effect of fibrinogen adsorption | suction suppression. These functional groups having a negative charge can be employed singly or in combination.

(Ligand having a negative charge with a hydrocarbon chain and / or a cyclic organic compound moiety and a charge of 0 or more)
Examples of ligands having a negative charge with a hydrocarbon chain and / or cyclic organic compound moiety and having a charge of 0 or more include phenylalanine, tryptophan, alanine, arginine, lysine, leucine, isoleucine, and histidine. From the viewpoint of ease of immobilization in the amino acid, amino acids are preferable, and phenylalanine and tryptophan are particularly preferable. In addition, when an amino acid becomes a ligand by forming a covalent bond with a carrier, the ligand has a structure changed from the original amino acid, but the state in which the amino acid is bound by condensation or the like is described in this specification. It is called “amino acid”.
For example, when the ligand is tryptophan, the indole ring moiety corresponds to a cyclic organic compound moiety, and the carboxyl group moiety corresponds to a negatively charged functional group.
Typically,
In the amino acid represented by the formula (1), the R (side chain) portion corresponds to a hydrocarbon chain and / or a cyclic organic compound portion, and CH ₃ in the case of alanine, and CH ₂ CH (CH ₃ ) _{2 in} the case of leucine. Is a chain. However, when the ligand is arginine, the side chain (R) is
However, since a molecular partial structure composed of carbon atoms and hydrogen atoms is called a hydrocarbon chain, the —CH ₂ —CH ₂ —CH ₂ — portion is a hydrocarbon chain.

(Histone)
Histone is a DNA-binding protein present in a biological fluid, and includes histone H1, histone H2A, histone H2B, histone H3, and histone H4. It is preferable to remove histone H3 and histone H4 from the inside, and it is particularly preferable to remove histone H3.

(Biological fluid)
The biological fluid includes all liquids derived from a biological body, and specifically includes body fluids such as blood, plasma, serum, and ascites, cell culture fluid, and the like.

(device)
FIG. 1 is a cross-sectional view showing an embodiment of a device. In FIG. 1, a device 100 is a module connected to a blood extracorporeal circuit. A cap 150 having an inlet port 140 is screw-fitted into an opening at one end of the cylinder 110 via a packing 130 with a filter 120 on the inside, and a filter 120 ′ is stretched on the inside at the other end opening of the cylinder 110. A cap 170 having an outlet port 160 is screwed through 130 ′ to form a container, and an adsorbent is filled and held in the gap between the filters 120 and 120 ′ to form a histone adsorbent layer 180.

  The histone adsorbent layer 180 may be filled with the adsorbent according to the present embodiment alone or in combination, and other carriers may be mixed or laminated. As other carriers, for example, adsorbents of other malignant substances such as cytokines, activated carbon having a wide range of adsorption ability, and the like can be used. Thereby, a wide range of clinical effects due to the synergistic effect of the carrier can be expected. Although the volume of the histone adsorbent layer 180 can be 5 mL to 3000 mL, 50 mL to 1000 mL is preferable, and 100 mL to 500 mL is more preferable from the viewpoint of the histone adsorption capacity and the extracorporeal blood volume during blood extracorporeal circulation.

  The module connected to the blood extracorporeal circuit is disposed in the blood extracorporeal circuit so as to come into contact with the biological fluid to be treated, and can be used for treating systemic inflammatory diseases.

  FIG. 2 is a cross-sectional view showing another embodiment of the device. In FIG. 2, the device 200 is a module that connects to an extracorporeal circuit. The carrier (hollow fiber) 220 is held by the potting agent 230 at both ends of the cylinder 210. Both end surfaces of the hollow fiber are open. A cap 250 having an inlet port 240 and a cap 250 ′ having an outlet port 260 are fitted into the cylinder 210, and the carrier (hollow fiber) 220 is filled and held. Further, a dialysate inlet 270 and a dialysate outlet 280 are provided on the side surface of the cylinder 210.

  When used in a blood purification method by extracorporeal blood circulation, blood derived from the body of the treatment target is introduced from the inlet port 240, passed through the hollow fiber-like adsorbent 220, and passed through the outlet port 260 through the body of the treatment target. The blood purification method by extracorporeal blood circulation can be performed by returning to the state. When the carrier is a hollow fiber, the dialysate is introduced from the dialysate inlet 270, the dialysate is led out from the dialysate outlet 280, and the dialysate in the device 200 is circulated. Dialysis can be performed during circulation. Further, the dialysate may not be circulated, and the dialysate inlet 270 and the dialysate outlet 280 may be opened. In this case, blood is filtered. Further, by blocking the dialysate inlet 270 and the dialysate outlet 280 with a stopper or the like, it is possible to suppress blood filtration and perform a blood purification method by extracorporeal blood circulation.

  The adsorbents and devices described above can be effectively used to suppress or treat organ inflammation in diseases mediated by histones. Specific diseases include sepsis, septic shock, toxic shock syndrome, ischemia reperfusion disorder, adult respiratory distress syndrome (ARDS), Paget's disease, osteoporosis, multiple myeloma, acute and chronic myelogenous leukemia, pancreas β-cell destruction, inflammatory bowel disease, psoriasis, Crohn's disease, ulcerative colitis, anaphylaxis, contact dermatitis, asthma, muscle degeneration, cachexia, Reiter syndrome, type I and type II diabetes, bone resorption, Examples include graft-versus-host reactions, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, fever, and muscle pain due to infection.

When the histone adsorbent according to this embodiment is brought into contact with a biological fluid or the like for a certain period of time, the histone adsorption rate before and after the treatment is calculated by the following equation.
Histone adsorption rate (%) = (C ₀ −C) / C ₀ × 100
C ₀ : histone concentration in the solution before treatment C: histone concentration in the solution after treatment

  In order to evaluate the histone adsorption rate of the adsorbent according to this embodiment, as a method of contacting with a biological fluid or the like, lipopolysaccharide (LPS) derived from Escherichia coli is added to blood collected from a healthy volunteer. Add, shake using a shaker, centrifuge using a centrifuge, obtain the supernatant as a plasma sample, and mix the obtained plasma sample with the adsorbent in a polypropylene (PP) tube. And a method of shaking using a shaker.

  In addition, E. coli was added to blood collected from healthy volunteers. To the adsorbent packed column created by adding the lipopolysaccharide derived from E.coli and shaking the blood using a shaker into a 2.5 mL laboratory column (Mobitech) with an adsorbent of 9 mm in inner diameter, The method of letting liquid flow using a syringe pump is mentioned.

  Furthermore, as a method of contacting with a full module of the same scale as the product, E. coli is applied to blood collected from healthy volunteers. Examples thereof include a method in which a lipopolysaccharide derived from E. coli is added and blood shaken using a shaker is circulated using a peristaltic pump to a device as shown in FIG. 1 filled with an adsorbent. At this time, in order to reduce the amount of blood used, it is preferable to reduce the capacity of the entire circulation circuit including the device.

  Hereinafter, although an example is given and explained concretely, the present invention is not limited to this at all.

<Example 1>
(Adsorbent preparation and evaluation)
Spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size 100 μm, exclusion limit molecular weight of about 1 million or more, amount of pullulan having a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 100 g In 1 L of dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 120 g of sodium hydroxide (Wako Pure Chemical Industries, Ltd.), 780 mL of epichlorohydrin (Wako Pure Chemical Industries, Ltd.), sodium borohydride (Wako Pure Chemical Industries, Ltd.) Epoxy group was introduced by reaction at 750 mg for 5 hours at 750 mg (manufactured by Yakuhin Co., Ltd.). After the reaction, it was washed with methanol (manufactured by Wako Pure Chemical Industries, Ltd.) and then washed with pure water to obtain an activated porous material. Next, 12 mg of tryptophan per 1 mL of the volume of the porous material in a wet state of the porous material is reacted in a pH 9.8 carbonate buffer solution to bind the amino group of tryptophan and the epoxy group of the porous material to thereby form the adsorbent A. Obtained. When the amount of immobilized tryptophan was calculated using elemental analysis, it was 55 μmol / mLgel.

(Plasma histone H3 adsorption test)
Escherichia coli 0127: B8-derived lipopolysaccharide (LPS) (manufactured by Sigma-Aldrich) was added to blood collected from healthy volunteers to a concentration of 0.1 μg / mL, and the mixture was shaken for 24 hours. And shaken at 39 ° C. Thereafter, the mixture was centrifuged at 2000 g for 10 minutes using a centrifuge, and the supernatant was obtained as a plasma sample. The obtained plasma sample (3 mL) and adsorbent A (0.5 mL) were mixed in a polypropylene (PP) tube and shaken at 39 ° C. for 1 hour using a shaker. The PP tube after shaking was centrifuged at 2000 g for 1 minute using a centrifuge, and the supernatant was obtained as a plasma sample after contacting the adsorbent.

(Histone removal performance evaluation)
Using the obtained plasma sample after contact with the adsorbent, the histone H3 adsorption rate after the treatment was calculated. The histone H3 adsorption rate calculation formula is shown below. The concentration of histone H3 was measured according to the attached instruction manual using EpiQuik (registered trademark) Total Histone H3 Quantification Kit (manufactured by EPIENTEK). The results are shown in Table 1.

<Example 2>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. Adsorbent B was obtained by reacting 6 mg of phenylalanine per 1 mL of the volume in the wet state of the porous material in a carbonate buffer solution of pH 9.8 to bind the amino group of phenylalanine and the epoxy group of the porous material. The amount of immobilization calculated using elemental analysis was 24 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent B.

<Example 3>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. In a carbonate buffer solution having a pH of 9.8, 12 mg of phenylalanine was reacted per 1 mL of the volume of the porous material in a wet state, and the amino group of phenylalanine and the epoxy group of the porous material were combined to obtain an adsorbent C. The amount of immobilization calculated using elemental analysis was 50 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent C.

<Example 4>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. In a pH 9.8 carbonate buffer solution, 12 mg of alanine was reacted per 1 mL of the volume of the porous body in a wet state, and the amino group of alanine and the epoxy group of the porous body were combined to obtain an adsorbent D. The amount of immobilization calculated using elemental analysis was 55 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent D.

<Example 5>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. In a carbonate buffer of pH 9.8, 12 mg of arginine was reacted per 1 mL of the volume of the porous material in a wet state, and the amino group of arginine and the epoxy group of the porous material were combined to obtain an adsorbent E. The amount of immobilization calculated using elemental analysis was 55 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent E.

<Example 6>
(Adsorbent preparation and evaluation)
A spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan with a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 1 g (equivalent to about 8 mLgel) was weighed, added to a falcon tube, and swollen with DMSO (10 mL). Thereto, methyl iodide (10 g, 70 mmol), sodium borohydride (5.0 mg) and aqueous sodium hydroxide solution (1.2 g / 1.8 mL) were added, and the mixture was reacted by shaking at 30 ° C. for 5 hours. . The resin was filtered and washed with MeOH followed by water to obtain adsorbent F. The amount of immobilization calculated using elemental analysis was 45 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent F.

<Example 7>
(Adsorbent preparation and evaluation)
A spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan with a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 1 g (equivalent to about 8 mLgel) was weighed, added to a falcon tube, and swollen with DMSO (10 mL). Thereto, methyl iodide (20 g, 140 mmol), sodium borohydride (10.0 mg), and aqueous sodium hydroxide solution (2.4 g / 3.6 mL) were added, and the reaction was shaken at 30 ° C. for 5 hours. The resin was filtered and washed with MeOH followed by water to obtain adsorbent G. The amount of immobilization calculated using elemental analysis was 81 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent G.

<Example 8>
(Adsorbent preparation and evaluation)
A spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan with a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 1 g (equivalent to about 8 mLgel) was weighed, added to a falcon tube, and swollen with DMSO (10 mL). Thereto, benzyl bromide (12 mL, 101 mmol), sodium borohydride (5.0 mg) and aqueous sodium hydroxide solution (1.2 g / 1.8 mL) were added and reacted by shaking at 30 ° C. for 5 hours. . The resin was filtered and washed with MeOH followed by water to obtain adsorbent H. The amount of immobilization calculated using elemental analysis was 34 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent H.

<Example 9>
(Adsorbent preparation and evaluation)
A spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan with a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 1 g (equivalent to about 8 mLgel) was weighed, added to a falcon tube, and swollen with DMSO (10 mL). Thereto, benzyl bromide (24 mL, 202 mmol), sodium borohydride (10.0 mg), aqueous sodium hydroxide solution (2.4 g / 3.6 mL) were added, and the reaction was shaken at 30 ° C. for 5 hours. The resin was filtered and washed with MeOH followed by water to obtain adsorbent I. The amount of immobilization calculated using elemental analysis was 63 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent I.

<Comparative Example 1>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. Add 2 mL of 10% sodium sulfite aqueous solution to 5 mL of activated porous body, and shake with a shaker at 80 ° C. for 2 hours to bind the sulfo group of sodium sulfite and the epoxy group of the porous body. Thus, an adsorbent J was obtained. The amount of immobilization calculated using elemental analysis was 20 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent J.

<Comparative Example 2>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. 4 mL of 10% sodium sulfite aqueous solution is added to 5 mL of the activated porous body, and the mixture is shaken with a shaker at 80 ° C. for 2 hours to bond the sulfo group of sodium sulfite and the epoxy group of the porous body. Thus, an adsorbent K was obtained. The amount of immobilization calculated using elemental analysis was 40 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent K.

<Comparative Example 3>
(Adsorbent preparation and evaluation)
In the same manner as in Example 1, an activated porous material was obtained. Add 9 mL of 10% sodium sulfite aqueous solution to 5 mL of activated porous body, and shake with a shaker at 80 ° C. for 2 hours to bind the sulfo group of sodium sulfite and the epoxy group of the porous body. Thus, an adsorbent L was obtained. The amount of immobilization calculated using elemental analysis was 88 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent L.

<Comparative example 4>
(Adsorbent preparation and evaluation)
A spherical porous body made of polyvinyl acetate (Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan with a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.20 mL / mL or more) 1 g (equivalent to about 8 mLgel) was weighed and swollen with DMSO (10 mL). Thereto, an aqueous chloroacetic acid solution (9.5 g / 10 mL), sodium borohydride (5.0 mg), an aqueous sodium hydroxide solution (1.2 g / 1.8 mL) were added, and the mixture was shaken at 30 ° C. for 5 hours. Adsorbent M was obtained. The amount of immobilization calculated using elemental analysis was 140 μmol / mLgel. Table 1 shows the results of the same adsorption test as in Example 1 using the adsorbent M.

  From the results of Table 1, the adsorbent supports A, B, C, D, E, F, G, H, in which the ligand has a hydrocarbon chain and / or a cyclic organic compound moiety, and the charge of the ligand is 0 or more. , I removes histone H3. In particular, in the adsorbent A characterized in that tryptophan is immobilized, histone H3 can be suitably removed. On the other hand, the adsorption carriers J, K, L, and M, whose ligands have only negative charges, did not remove histone H3 at all.

  The adsorption carrier and the biological fluid purification device of the present invention can efficiently remove, for example, histone H3 from biological fluid, and are useful for the treatment of diseases that develop or worsen through histones typified by SIRS. .

  This biological fluid purification device can be used for treatment of SIRS caused by histones. By bringing the adsorbent of the present invention into contact with a biological fluid, histones in the biological fluid can be efficiently adsorbed to the absorbent, and the histones can be removed from the biological fluid. Since histones can be removed, it can be effectively used to suppress or treat systemic inflammation in diseases mediated by histones. Specific diseases include sepsis, septic shock, toxic shock syndrome, ischemia reperfusion disorder, adult respiratory distress syndrome (ARDS), Paget's disease, osteoporosis, multiple myeloma, acute and chronic myelogenous leukemia, pancreas β-cell destruction, inflammatory bowel disease, psoriasis, Crohn's disease, ulcerative colitis, anaphylaxis, contact dermatitis, asthma, muscle degeneration, cachexia, Reiter syndrome, type I and type II diabetes, bone resorption, Examples include graft-versus-host reactions, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, fever, and muscle pain due to infection. In particular, it is suitably used for the purpose of improving the pathological condition of systemic inflammatory response syndrome (SIRS) such as sepsis.

  Histone has been reported to have cytotoxicity, and this biological fluid purification device can be suitably used for the purpose of removing histone having cytotoxicity from a cell culture solution. Of course, the industrially applicable fields of the present invention are not limited to the above.

  100, 200 ... device, 110, 210 ... cylinder, 120, 120 '... filter, 130, 130' ... packing, 140, 240 ... inlet port, 150, 170, 250, 250 '... cap, 160, 260 ... outlet port , 180 ... organ inflammation suppressing carrier layer, 220 ... carrier (adsorption carrier), 230 ... potting agent, 270 ... dialysate inlet, 280 ... dialysate outlet.

Claims (8)

  An adsorbent that removes histone from a biological fluid, wherein a ligand is immobilized on a water-insoluble carrier, the ligand has a carbon hydrogen chain and / or a cyclic organic compound moiety, and the charge of the ligand Is an adsorbent with 0 or more.   The adsorbent according to claim 1, wherein the charge of the ligand is zero.   The adsorbent according to claim 1 or 2, wherein the ligand includes a functional group having a negative charge.   The adsorbent according to any one of claims 1 to 3, wherein the ligand is an amino acid.   The adsorbent according to any one of claims 1 to 4, wherein the amino acid is tryptophan or phenylalanine.   The adsorbent according to any one of claims 1 to 5, wherein the water-insoluble carrier is a porous bead carrier made of polyvinyl alcohol gel.   The adsorbent according to any one of claims 1 to 6, wherein the histone is histone H3.   A biological fluid purification device for removing histone from biological fluid, wherein the device has a housing provided with an inlet and an outlet for the biological fluid, and is housed in the housing. A biological fluid-purifying device that removes the histone from the biological fluid when the biological fluid flows in the housing.