JPH09313604A - Chemokine adsorbent, adsorption removal method and adsorber - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to efficiently adsorb and remove various of the chemokines (exclusive of interleukin-8) existing in humor by fixing a compd. having logP of a prescribed value or above to a water-insoluble carrier. SOLUTION: The adsorptive material of the chemokines (exclusive of interleukin-8) in the humor is formed by fixing the compd. having the logP (P is the distribution coefft. in an octanol water system) value of >=2.50 to the water-insoluble carrier. In such a case, the chemokines refer to materials having chemotaxis. The chemokines of which the genes coding these materials belong to a CXC sub-family exist in the human 4th chromosomes (q12 to 21) and the chemokines belonging to a CC sub-family exist in the human 17 the chromosomes (q11 to 12). The adsorption of the chemokines on the adsorptive material is conceivably achieved by the hydrophobic interaction between the atom groups introduced onto the carrier and the chemokines by the fixation of the compd. having the logP value of >=2.50.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an adsorbent for adsorbing and removing chemokines in body fluids, a method for adsorbing and removing chemokines in body fluids using the adsorbents, and a method for adsorbing and removing chemokines from body fluids. Chemokine adsorber.

[0002]

BACKGROUND OF THE INVENTION Immunocompetent cells produce various active substances when they elicit an immune response. Some of them are proteinaceous substances called cytokines and play a very important role as biological defense factors deeply involved in various antigen-specific and non-specific immune inflammatory reactions. Although cytokines are essentially indispensable for maintaining homeostasis in the living body, they are excessively produced in pathological conditions such as inflammation and are involved in the pathogenesis and prolongation of inflammation.

Among cytokines, those having chemotaxis are particularly chemokines (Chemokin).
e) is collectively called. Chemotaxis is also called chemotaxis, and refers to chemotaxis that is stimulated by the difference in concentration of chemical substances.
A substance called a chemokine is characterized by its structural characteristics.
Known to form two families.

The characteristics of chemokines are about 6,000.
It exists mainly as a protein having a molecular weight of 1 to 10,000, but depending on the type of chemokine,
Some of them exist in a state in which the molecular weight is larger than the above range due to formation of a dimer or tetramer in a solution or O-glycosylation. Chemokines are classified into the following two subfamilies based on their structural characteristics. In other words, M. Baggiolini et al. Introduced the review "CC CHEMOKINES IN ALLERGIC
INFLAMMATION) ", Immunology Today (Immunolo
gy Today), Vol. 15, p. 127, 1994, chemokines have four cysteine residues (hereinafter C) at very conserved positions in the molecule, The four Cs are C1, C2, C3,
C4 is a CXC subfamily in which one arbitrary amino acid (hereinafter referred to as X) exists between C1 and C2, and CC in which no amino acid exists between C1 and C2.
It is classified into subfamilies. Furthermore, chemokines within each subfamily have been shown to have homology in amino acid sequences other than C (eg, Chihara's report, Clinical Immunity, Vol. 27 [Sup].
pl. 16], pp. 162-171, 1995).

Among the leukocytes, the CXC subfamily generally acts on neutrophils, and the CC subfamily is said to generally act on monocytes, eosinophils, basophils and lymphocytes. It has been suggested that the effects are diverse. For example, interleukin-8 is an interleukin having chemotaxis among interleukins and is a chemokine classified into the CXC subfamily. Its function is not only neutrophils but also lymphocytes.
It is known that basophils, eosinophils, cutaneous keratinocytes, melanoma cells, fibroblasts, and vascular endothelial cells also show their biological activity (Matsushima, Clinical Immunity, Vol. 27 [Supp.
l. 16], pp. 147-154, 1995).

[0006] For example, on human monocytes, monocy, which is one of the chemokines classified into the CC subfamily,
te chemoattractant protein-1 (hereinafter referred to as MCP-1) and macrophage inflammatory protein-1 (macropha
In addition to the presence of specific receptors for ge inflammatory protein-1 (hereinafter, referred to as MIP-1), it is possible to treat three types of chemokines classified into CC subfamily of MCP-1, MIP-1, and RANTES. It has been suggested that a common receptor exists, and it is known that some subfamilies exhibit the same physiological activity through the same receptor (Matsushima, Clinical Immunity). 27, [Suppl. 16], 1
47-154, 1995).

When a living body is invaded from the outside, inflammation as a biological defense reaction is caused and leukocyte infiltration occurs in the inflamed area. Leukocyte infiltration into such an inflamed area is
It is caused by the leukocyte chemotactic factor produced at the site of inflammation. It is known that a chemokine plays a role as a factor that causes the infiltration of leukocytes.
In fact, one of the chemokines in the rabbit acute inflammation model
It has been demonstrated that administration of an antibody against interleukin-8 (anti-interleukin-8 antibody), which is one of the two, blocks neutrophil infiltration and prevents organ damage associated with acute inflammation (Sekido et al., Nature
e), 365, pp. 654-657, 1993).

More recently, systemic inflammatory response syndrome (syst
emic inflammatory response syndrome (SIRS))
In the pathological condition encompassed by the concept, excessive production of various cytokines activates the cytokine network, which in turn leads to excessive production of chemokines, which induces and activates neutrophils. (Endo et al., Intensive care, 4th
Vol., Pp. 1357-1365, 1992), and it is suggested that a systemic inflammatory reaction progresses with it, causing shock, tissue damage and multiple organ failure, and eventually dying.

RANTES, platelet factor- (platelet factor-
4 (hereinafter referred to as PF-4)) and macrophage inflammatory protein-1α (macrophage inflammatory protein-1)
It has been suggested that various inflammatory cells such as lymphocytes and eosinophils are infiltrated by the action centered on chemokines such as α (hereinafter referred to as MIP-1α).

Further, for example, contact with an artificial material during extracorporeal blood circulation such as dialysis therapy, stimulating substances represented by endotoxin in dialysate, various stimulating factors existing in blood or tissue, etc. It has been pointed out that stimulation of immunocompetent cells may result in excessive production of chemokines,
For example, in dialysis amyloidosis and carpal tunnel syndrome, which are complications associated with long-term dialysis therapy, MCP-1 and MIP-1
It has been suggested that α is overproduced and may be involved in the pathogenesis (Inoue et al., Nephology dialysis transplantation (Nephro).
logy DialysisTransplantation), Volume 10, 2077
Pp. 2082, 1995).

Further, gouty arthritis, psoriasis, contact dermatitis, idiopathic pulmonary fibrosis, adult respiratory distress syndrome, inflammatory bowel disease, immune vasculitis, urinary tract infection, myocardial infarction, asthma, respiratory tract infection, In diseases such as perinatal infection and transplant organ rejection, one of the chemokines, interleukin-8, has been detected in abnormally high concentrations in local inflammation or systemic blood compared to normal individuals (immunity. Pharmacology, Volume 12, Issue 1, 15
-21, 1994).

In rheumatoid arthritis, interleukin-8, RANTES, MCP-1, MIP-
1α and macrophage inflammatory protein-1β (macroph
ageinflammatory protein-1β (hereinafter referred to as MIP-1β) is MCP-1 in crescentic nephritis,
It was suggested that MIP-1α and MIP-1β are abnormally expressed in interleukin-8 and MCP-1 in chronic glomerulonephritis, and MCP-1 is abnormally expressed in lupus nephritis, and are involved in the pathogenesis thereof. There is.

As a method for removing chemokines in body fluids having such various functions, almost no report has been found so far, but if it mentions, it is positive in the surface of JP-A-6-312017. A method for purifying blood with an endotoxin comprising a porous carrier having a functional group and / or an adsorbent for a cytokine derived from the endotoxin is disclosed. However, nothing is disclosed in the examples with respect to cytokine measurement or cytokine adsorption.

It is also possible to apply so-called anti-cytokine therapy in which the action of chemokines is suppressed by administering an antibody against chemokines or a substance that inhibits the binding of chemokines to receptors. However, it has been suggested that various types of chemokines are abnormally expressed in pathological conditions involving chronic inflammation such as rheumatoid arthritis, and in order to suppress these effects by administration of antibodies and the like, It is necessary to prepare and administer an antibody against each. Further, the antibody or the like to be administered should not adversely affect the human body, and it is considered that it takes a long time and a large amount of cost to develop it, and thus it cannot be said to be an appropriate therapeutic method.

[0015]

The present invention provides an adsorbent capable of efficiently adsorbing and removing various chemokines present in body fluids, a method for adsorbing and removing chemokine using the adsorbent, and the adsorbent. The purpose is to develop the chemokine adsorber used.

[0016]

[Means for Solving the Problems] The present inventors have made extensive studies on an adsorbent capable of efficiently adsorbing and removing chemokines present in body fluids. As a result, log in water-insoluble carrier
P (P is partition coefficient in octanol-water system) value is 2.5
The present invention has been completed by discovering that a substance having 0 or more compounds immobilized can efficiently adsorb and remove chemokines present in body fluids.

The applicant of the present invention uses a water-insoluble carrier as a logarithm.
P (P is partition coefficient in octanol-water system) value is 2.5
An application for an adsorbent of interleukins, in which 0 or more compounds are immobilized, has been previously filed (Japanese Patent Application No. 7-6656).
No. 5) excludes interleukin-8 (chemokines belonging to the CXC subfamily) having chemotaxis among the interleukins in the present invention.

That is, according to the present invention, (1) a compound having a log P (P is a partition coefficient in an octanol-water system) value of 2.50 or more is immobilized on a water-insoluble carrier. However, it relates to an adsorbent of (excluding interleukin-8).

Further, the present invention relates to (2) an adsorbent for a chemokine (excluding interleukin-8) in a body fluid according to item (1), wherein the water-insoluble carrier is a hydrophilic carrier.

The present invention further provides (3) the chemokine in body fluid according to the above (1) or (2), wherein the water-insoluble carrier has a porous structure (excluding interleukin-8).
Adsorbent.

The present invention further comprises (4) a water-insoluble carrier,
The present invention relates to an adsorbent for a chemokine (excluding interleukin-8) in body fluid according to the above (1), (2) or (3), wherein the exclusion limit molecular weight of the globular protein is 10,000 or more and 600,000 or less.

The present invention further provides (5) the chemokine according to item (1) above (excluding interleukin-8).
Chemokines in body fluids (excluding interleukin-8), characterized in that the body fluid is brought into contact with the adsorbent of
The adsorption removal method of

Further, the present invention provides (6) a chemokine according to the above item (1) in a container having a liquid inlet and an outlet and provided with a means for preventing the adsorbent from flowing out of the container (however,
It relates to an adsorber for chemokines (excluding interleukin-8) in body fluids, which is characterized by being filled with an adsorbent for interleukin-8.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The body fluid in the present invention is blood,
A liquid component derived from a living body such as plasma, serum, ascites, lymph, and joint fluid.

The chemokine in the present invention is a substance having chemotaxis, and the gene encoding the substance is a chemokine belonging to the CXC subfamily on human chromosome 4 (q12 to 21), and CC sub Chemokines belonging to the family are human chromosome 17 (q11-1
2) A substance characterized by being present in 2). However, interleukin-8 is excluded. Report by Matsushima (Clinical Immunity, Vol. 27 [Suppl. 16], 147-154.
Page, 1995) and Chihara's report (Clinical Immunity, Vol. 27 [Supp. 16], 162-171, 19).
(See 1995), the human chemokines known to date are enumerated as GROα, GROβ, GROγ, as those classified into the CXC subfamily.
Neutrophil activating protein-2
ein-2 (NAP-2)), neutrophil activating protein-4 (N
AP-4), epithelial-cell derived neutrophil-act
ivating protein-78 (ENA-78), PF-4, inte
rferon-inducible protein-10 (IP-10), granulo
cyte chemotactic protein-2 (GCP-2), β-thromboglobulin (βTG) and pre-B cell growth stimulat
ing factor (PBSF). In addition, MCP-1, H is classified into the CC subfamily.
C14, monocyte chemoattractant protein-3 (MCP
-3), I-309, MIP-1α, MIP-1β, R
Antes is given. However, the names of chemokines may not be unified, and even the same substance may be referred to by different names. For example, GROβ and GROγ are macrophage inflammatory protein-2α, respectively.
(MIP-2α)), macrophage inflammatory protein-2
β (macrophage inflammatory protein-2β (MIP-
2β)), and MCP-1 is also a monocyte chemotactic and activating factor.
(MCAF)), HC14 is a monocyte chemo
Also known as attractant protein-2 (MCP-2), a book edited by the Department of Microbiology, Kyoto Prefectural University of Medicine ("Cytokine Data Manual", Nankodo, 1
995). Therefore, it goes without saying that the above chemokines are also included as chemokines when called by other names. Furthermore, it goes without saying that it also includes substances that are newly discovered in the future and are recognized to fall within the definition of chemokine.

The chemokine (except interleukin-8, hereinafter the same) adsorbent of the present invention is characterized in that a compound having a logP value of 2.50 or more is immobilized on a water-insoluble carrier. is there.

The log P value becomes a parameter of the hydrophobicity of the compound, and the partition coefficient P in a typical octanol-water system.
The method of obtaining is as follows. First, dissolve the compound in octanol (or water), add an equal amount of water (or octanol), and add Griffin Flask.
Shaker (Griffin flask shaker)
And George Limited (Griffin & George Lt
D.)) for 30 minutes. After that, 2,000r
It is determined by the following formula by centrifuging at pm for 1 to 2 hours and measuring the compound concentration in the octanol layer and the aqueous layer by various methods such as spectroscopic analysis or GLC.

P = Coct / Cw Coct: Concentration of compound in octanol layer Cw: Concentration of compound in water layer Logs of various compounds by many researchers so far.
P-values have been measured, but those measured values are organized by C. Hansch and others ("PARTITION COEFFICIENTS AND THEIR USES", Chemical Review). (Chemical Reviews), Vol. 71, 5
(See page 25, 1971).

For compounds whose measured values are unknown, RF Rekker wrote in his book (“THE HYDROPHOBIC FRAGMENTAL CONSTAN”).
T) ", Elsevier Scientific Publishing Company (Elsevier Sci. Pub.Com.), Amsterdam (1977)) and the calculated value (?)
f) is helpful. The hydrophobic fragment constant is a value showing the hydrophobicity of various fragments determined by performing statistical processing based on a large number of logP measured values, and the sum of the f values of each fragment constituting the compound is lo.
It is reported to be in good agreement with the gP value. In the present invention, the term “log P value” means the Σf value for a compound whose log P value is unknown.

In searching for a compound effective for chemokine adsorption, compounds having various logP values were fixed and examined. As a result, the logP value was 2.50 or more, preferably 2.7.
It was found that compounds of 0 or more, more preferably 2.90 or more are effective for chemokine adsorption, and compounds having a logP value of less than 2.50 show almost no chemokine adsorption ability. For example, when an alkylamine is immobilized, the alkylamine is converted into n-hexylamine (logP = 2.0).
It was found that when 6) was changed to n-octylamine (logP = 2.90), the chemokine adsorption capacity was dramatically increased during this period. From these results, it is considered that the chemokine adsorption to the adsorbent of the present invention is due to the hydrophobic interaction between the chemokine and the atomic group introduced on the carrier by immobilizing the compound having a logP value of 2.50 or more. It is considered that a compound having a logP value of less than 2.50 does not exhibit chemokine adsorption ability because its hydrophobicity is too small.

On the other hand, when n-octylamine is changed to cetylamine (Σf = 7.22), which has a longer alkyl chain length and is considered to have higher hydrophobicity, chemokine adsorption capacity can be further increased. all right. From these results, it is found that the chemokine adsorption on the chemokine adsorbent of the present invention is achieved by immobilizing the compound having a logP value of 2.50 or more. However, the larger the logP value of the compound is, the better it is, for example, cetylamine. By immobilizing a compound such as octadecylamine (Σf = 8.28), which is considered to have a longer alkyl chain length and higher hydrophobicity, it shows chemokine adsorption equivalent to or higher than that when cetylamine is immobilized. It is considered to be a thing. The upper limit of the logP value is not particularly limited, but is practically about 15.

In the present invention, as the compound fixed to the water-insoluble carrier, any compound having a logP value of 2.50 or more can be used without particular limitation. However, when the compound is bound to the carrier by the chemical bonding method, a part of the compound is often eliminated, but when the leaving group greatly contributes to the hydrophobicity of the compound, that is, When the hydrophobicity of the atomic group fixed on the carrier by desorption becomes smaller than Σf = 2.50, it is unsuitable as the compound used in the present invention in view of the gist of the present invention. As a representative example thereof, there is a case where isopentyl benzoate (Σf = 4.15) is immobilized on a carrier having a hydroxyl group by transesterification. In this case, the atomic group actually fixed on the carrier is C
₆ H ₅ CO-, and Σf of this atomic group is 1 or less.
Whether such a compound is suitable as a compound to be used in the present invention depends on whether the leaving group is replaced by hydrogen.
It may be determined based on whether the ogP value is 2.50 or more.

Among the compounds having a log P value of 2.50 or more, those having a hydrocarbon moiety having 7 to 20 carbon atoms, especially 8 to 18 carbon atoms are preferable, and further unsaturated hydrocarbons, alcohols, amines, thiols, Compounds having a functional group that can be used for binding to a carrier, such as carboxylic acid and its derivatives, halides, aldehydes, isocyanates, oxirane ring-containing compounds such as glycidyl ether, and halogenated silanes are preferable.

As the unsaturated hydrocarbon, 1-heptene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, 1
-Examples include Eikosen.

Examples of the alcohol include n-octyl alcohol, dodecyl alcohol, hexadecyl alcohol, 1-octen-3-ol, naphthol, diphenylmethanol and 4-phenyl-butanol.

Examples of the amine include n-octylamine, decylamine, dodecylamine, hexadecylamine, octadecylamine, naphthylamine, 2-aminooctene, diphenylmethylamine and the like.

Examples of the thiol include octanethiol, decanethiol, dodecanethiol, tetradecanethiol, hexadecanethiol and octadecanethiol.

Examples of the carboxylic acid and its derivative include n-octanoic acid, nonanoic acid, 2-nonenoic acid, decanoic acid, dodecanoic acid, stearic acid, arachidonic acid, oleic acid, diphenylacetic acid, and the like. Examples thereof include acid halides, esters, amides and hydrazides of the above carboxylic acids.

Examples of the halide include octyl chloride, octyl bromide, decyl chloride and dodecyl chloride.

Examples of the aldehyde include octyl aldehyde, n-caprin aldehyde and dodecyl aldehyde.

Examples of the above-mentioned isocyanates include dodecyl isocyanate, hexadecyl isocyanate, octadecyl isocyanate and the like.

Examples of the glycidyl ether include dodecyl glycidyl ether, hexadecyl glycidyl ether, octadecyl glycidyl ether and the like.

Examples of the halogenated silane include n-octyltrichlorosilane and octadecyltrichlorosilane.

In addition to these compounds, compounds in which the hydrogen atom of the hydrocarbon moiety of the above-exemplified compounds is substituted with a substituent containing a hetero atom such as halogen, nitrogen, oxygen and sulfur, and other alkyl groups, etc. Of which, the logP value is 2.5
Zero or more compounds, the review of C. Hansch et al., “Partition Coeficients and
There Youths (PARTITION COEFFICIENTS AND THEIR U
SES ”, Chemical Reviews
s), vol. 71, p. 525, and the log P value shown in the table of p. 555 to p. 613 in 1971 is 2.50.
The above compounds and the like can be used, but the present invention is not limited thereto.

These compounds may be used alone, in any combination of two or more kinds, and may be used in combination with a compound having a logP value of less than 2.50.

The water-insoluble carrier in the adsorbent of the present invention means that it is solid and water-insoluble at normal temperature and pressure. The shape of the water-insoluble carrier in the present invention may be granular, plate-like, fiber-like, hollow fiber-like or the like, but its shape is not particularly limited and its size is not particularly limited.

As the water-insoluble carrier in the adsorbent of the present invention, glass beads, inorganic carriers such as silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide and crosslinked polystyrene, celluloses, crosslinked agarose, Representative examples are organic carriers composed of polysaccharides such as cross-linked dextrin, and composite carriers such as organic-organic and organic-inorganic obtained by combining these.

Among them, the hydrophilic carrier is preferable because the non-specific adsorption is relatively small and the chemokine adsorption selectivity is good. The hydrophilic carrier as used herein refers to a carrier having a contact angle with water of 60 degrees or less when a substance constituting the carrier is formed into a flat plate. Various methods for measuring the contact angle of water are known, but for example, Ikeda's book ("Selection of Experimental Chemistry / Colloid Chemistry, Chapter 4, Thermodynamics of Interfaces", Yukabo, 75-1)
As shown in p. 04, 1986), the most general method is to place a water drop on a flat plate of the substance to be measured. As the carrier having a contact angle of water of 60 degrees or less measured by the above method, cellulose, polyvinyl alcohol,
Representative examples are carriers made of saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethylmethacrylate, polyacrylic acid-grafted polyethylene, polyacrylamide-grafted polyethylene and glass. .

In addition, in the above-mentioned cellulose, the contact angle of water is shown to be about 18 degrees in the textbook (Rui Yoshito, "Medical Polymer Materials", Kyoritsu Shuppan, p. 65, 1989).
See year).

It is more preferable that these water-insoluble carriers have a large number of pores of an appropriate size, that is, a carrier having a porous structure. A carrier having a porous structure is, of course, a carrier having a space (macropore) formed by agglomeration of microspheres when the basic polymer matrix forms one spherical particle by agglomeration of microspheres. However, it has a three-dimensional structure (polymer network) in the case of a carrier having pores formed between the agglomerates of nuclei in one microsphere that constitutes the basic polymer matrix. Also included is a carrier having pores (micropores) present when the copolymer is swollen in a miscible organic solvent.

Considering the adsorption capacity per unit volume of the adsorbent, the water-insoluble carrier having a porous structure is preferably totally porous rather than surface porous, and the pore volume and specific surface area are It is preferably large enough not to impair the adsorptivity.

As a carrier satisfying these preferable requirements, a porous gel made of cellulose can be mentioned. Porous gels made of celluloses (1) have relatively high mechanical strength and are tough so that they are less likely to be broken or produce fine powder by an operation such as stirring. Since it does not consolidate even at high speed, it can flow at a high flow rate, and its pore structure is not easily changed by high-pressure steam sterilization. (2) Since the gel is composed of celluloses, it is hydrophilic. Yes, there are many hydroxyl groups that can be used for ligand binding, and there is little non-specific adsorption. (3) Even if the pore volume is increased, the strength is relatively high, so an adsorption capacity that does not fit in a soft gel can be obtained. ,
(4) It has an excellent point that safety is higher than that of synthetic polymer gel and the like, and it is one of the most suitable carriers used in the present invention.

The term "celluloses" as used in the present invention means at least one of natural cellulose, regenerated cellulose and cellulose derivative. Examples of natural cellulose include degreased cotton fibers, hemp fibers, and lignin from wood. Pulp obtained by removing hemicellulose etc.,
There is a refined cellulose obtained by further refining the pulp. In addition, regenerated cellulose is cellulose obtained by once converting natural cellulose into a cellulose derivative and then regenerating it by hydrolysis or the like. Examples of the cellulose derivative include those obtained by esterifying and / or etherifying some or all of the hydroxyl groups of natural or regenerated cellulose. Specific examples of those in which a part or all of the hydroxyl groups of the cellulose are esterified, for example, cellulose acetate, cellulose propionate, cellulose butyrate, nitrocellulose, cellulose sulfate, cellulose phosphate, cellulose acetate butyrate, cellulose nitrate, of cellulose. Examples thereof include, but are not limited to, dicarboxylic acid esters. Specific examples of those in which a part or all of the hydroxyl groups of the cellulose are etherified include, but are not limited to, methyl cellulose, ethyl cellulose, benzyl cellulose, cyanoethyl cellulose, carboxymethyl cellulose, aminoethyl cellulose, oxyethyl cellulose and the like. Not something.

In the present invention, each of the above-mentioned carriers may be used alone, or any two or more of them may be mixed and used.

Further, the water-insoluble carrier having a porous structure as described above has a characteristic that the substance to be adsorbed can invade into the pores with a certain high probability, but invasion of other proteins does not occur as much as possible. More preferable. That is, the chemokine, which is an object to be adsorbed by the adsorbent of the present invention, often exists as a protein having a molecular weight of about 6,000 to 10,000, and in order to efficiently adsorb this protein,
Chemokines can invade into the pores with a certain probability, but it is preferable that other proteins do not invade as much as possible. The exclusion limit molecular weight is generally used as a measure of the molecular weight of a substance that can enter the pores. Exclusion limit molecular weight refers to written books (for example, Hiroyuki Hatano, Toshihiko Hanai,
As described in "Experimental High Performance Liquid Chromatograph", Kagaku Dojin, etc., it refers to the molecular weight of the molecule having the smallest molecular weight among the molecules that cannot penetrate (exclude) into the pores in gel permeation chromatography. The exclusion limit molecular weight is generally well investigated for globular proteins, dextran, polyethylene glycol and the like, but in the case of the carrier used in the present invention, it is appropriate to use the value obtained by using globular proteins.

As a result of examination using carriers having various exclusion limit molecular weights, it was revealed that the exclusion limit molecular weight range of the globular protein suitable for chemokine adsorption is 10,000 or more and 600,000 or less. That is, when a carrier in which the exclusion limit molecular weight of globular protein is less than 10,000 is used, the chemokine adsorption amount is small and its practicality decreases, and when it exceeds 600,000, proteins other than chemokines (mainly Adsorption of albumin) is increased, and its practicality is reduced in terms of selectivity. Therefore, the preferred range of exclusion limit molecular weight of the globular protein of the carrier used in the present invention is 10,000 or more 60
10,000 or less, more preferably 20,000 or more and 500,000 or less, and particularly preferably 30,000 or more and 400,000 or less.

Furthermore, it is preferable that the carrier has a functional group that can be used in the immobilization reaction of the ligand. Representative examples of these functional groups include hydroxyl group, amino group, aldehyde group, carboxyl group, thiol group, silanol group, amide group, epoxy group, halogen group, succinimide group, acid anhydride group, and tresyl group. , But is not limited to these.

As the carrier to be used in the present invention, either a hard carrier or a soft carrier can be used, but when it is used as an adsorbent for extracorporeal circulation, it can be packed into a column and passed through a liquid. It's important not to clog,
For that purpose, sufficient mechanical strength is required. Therefore, the carrier used in the present invention is more preferably a hard carrier. The term “hard carrier” as used herein means, for example, in the case of a granular gel, as shown in the reference example described later, the relationship between the pressure loss ΔP and the flow velocity when the gel is uniformly packed in a cylindrical column and an aqueous fluid is flown is 0. A linear relationship up to 3 kg / cm ² .

The adsorbent of the present invention has a logP value of 2.50.
The above compounds can be obtained by immobilizing them on a water-insoluble carrier, and various known immobilization methods can be used without particular limitation. However, when the adsorbent of the present invention is used for extracorporeal circulation treatment, it is important for safety to suppress desorption and elution of ligand during sterilization or treatment, and for that purpose, it is immobilized by a covalent bond method. Preferably.

The adsorbent used in the present invention is logP.
It is preferable that an appropriate amount of a compound having a value of 2.50 or more is immobilized. If the fixed amount is too small, chemokine is not adsorbed, and if the fixed amount is too large, stickiness such as platelets may occur when blood is used as the body fluid. Therefore, log
The fixed amount of the compound having a P value of 2.50 or more is preferably 10 to 1000 μm per dry weight (1 g) of the water-insoluble carrier.
mol, and more preferably 50 to 500 μmol
And most preferably 100 to 300 μmol.

As the adsorbent of the present invention, celluloses having a compound having a logP value of 2.50 or more immobilized (hereinafter,
It is preferable that the hydrogel has a colloidal solid phase composed of C-celluloses) and water.
The weight ratio of cellulose to water is preferably in the range of 1: 9 to 3: 7. The adsorbent will be described below.

The logP value is 2.5 with respect to celluloses.
As a method for immobilizing 0 or more compounds, various known methods such as a method of physically binding, a method of binding via an ionic bond, a method of binding via a covalent bond, etc. can be used. Since it is important that the bound compound is hard to be released, it is most preferable to immobilize it via a covalent bond. As a specific means, a method of directly bonding the compound through an ester bond, an amide bond, an ether bond, a thioether bond, a urethane bond or the like using a hydroxyl group of celluloses, or an amino group to celluloses, A method in which a functional group such as an aldehyde group, an epoxy group, or a carboxyl group is introduced to make the compound highly reactive (activated), and then the compound is bound can be mentioned. Further, as a specific method for introducing a functional group into cellulose to activate it, cyanogen bromide method, epoxy method, tresyl chloride property, periodate oxidation method and the like can be mentioned as specific examples. It is not limited to.

The hydrogel referred to in the present invention means a colloidal solid phase containing water or an aqueous solution as an essential component as described above, and the gel skeleton left after drying the hydrogel to remove water, that is, the xerogel is the main component. This is outside the concept of hydrogel in the invention.

The weight ratio of C-cellulose to water is the weight (wet weight W) when water attached to the hydrogel or interstitial water between the hydrogel particles is removed by a suction filtration method or a centrifugal method.
w) and the weight (dry weight Wd) when the weight is no longer changed by subsequent drying. That is,
Wd: (Ww-Wd) is the weight ratio of C-cellulose and water in the present invention. As a specific method for obtaining the wet weight, for example, take a hydrogel in a glass filter,
There is a method in which suction is performed using an aspirator and the weight is measured after the elapse of the suction time when the weight decrease curve showing the relationship between the suction time and the weight is created and the slope of the curve becomes gentle. Further, the drying method is not particularly limited as long as constant weight can be achieved, but atmospheric pressure drying at a temperature of about 105 ° C. can be simply adopted without a special device. Although the weight ratio of C-cellulose and water constituting the hydrogel of the present invention is in the range of 1: 9 to 3: 7, this weight ratio of the hydrogel is due to the pressure loss when the body fluid is passed through the chemokine adsorber. From the viewpoint of preventing deformation and the resulting consolidation, the ratio of C-cellulose is preferably 1/9 or more, and from the viewpoint of preventing the reduction of the purification rate, the ratio of C-cellulose is 3/7. Based on the following being desirable:

The spherical hydrogel particles housed in the chemokine adsorber of the present invention, particularly the hydrogel particles housed in the chemokine adsorber used as a blood purifier, preferably have a spherical shape, and the spherical shape means a spherical shape. Of course, it includes a wide range of spheroids. The average particle size of this type of hydrogel is generally 30 when treating a body fluid containing almost no cellular components such as plasma.
Those having a particle diameter of 0 μm or less are adopted from the viewpoint of high purification rate, and when used in the direct blood perfusion method, 250 to 100 to secure a sufficient passage channel for blood cell components.
Those of 0 μm are preferably used. The spherical hydrogel particles of the present invention, particularly the spherical hydrogel particles accommodated in the chemokine adsorber used as a blood purifier, preferably have a particle size of 300 to 600 μm from the viewpoint of compatibility between the permeability of blood cell components and the purification rate. The average particle size as used herein refers to the number average particle size, for example, with a stereoscopic microscope at a magnification of about 10 to 50 times,
It is determined by observing and measuring the particle size of 50 hydrogel particles and averaging the measured values. When the hydrogel particles are spheroids, the major axis is the particle size,
The average particle size is determined by the same method as described above. If the particle size is within the above range, the particle size distribution can be used without any particular limitation. For example, JP-A-63-117
Particles having a narrow particle size distribution, which are produced by forming uniform droplets using the vibration method described in Japanese Patent No. 039, etc. and coagulating under appropriate conditions, are particularly preferable when used in the direct blood perfusion method.

As a method for producing hydrogel particles composed of C-cellulose of the present invention, the cellulose is treated with C-cellulose.
Either a method of forming celluloses and then forming them into hydrogel particles, or a method of preparing hydrogel particles of celluloses and immobilizing a compound having a logP value of 2.50 or more thereto is possible.

More specifically, a method of forming hydrogel particles by forming celluloses into C-celluloses and then forming them into hydrogel particles will be described. C-celluloses were prepared by fixing compounds having a logP value of 2.50 or more to celluloses. After that, the C-celluloses are dissolved in a solvent to form a solution, and then the droplets are solidified to form hydrogel particles. If there is no suitable solvent for forming the C-celluloses into a solution, C is used. Examples of the method include, but are not limited to, a method in which cellulose is further esterified or etherified to make it easily soluble in a solvent, and then hydrogel particles are formed.

In the method of preparing hydrogel particles of cellulose and immobilizing the compound having a logP value of 2.50 or more, the method of preparing hydrogel particles of cellulose is as follows. There is a method of forming hydrogel particles by dropletizing and coagulating, and a method of using a cellulose derivative as the celluloses is preferably used because the selection range of the production conditions is widened due to the variety of types of solvents. Examples of the cellulose derivative include those in which a part or all of the aforementioned hydroxyl groups of cellulose have been esterified and / or etherified. Among them, cellulose acetates such as cellulose acetate and cellulose propionate are soluble in various solvents. Therefore, it is more preferably used. After these cellulose derivatives are hydrogel particles, they are hydrolyzed as they are or, if necessary, after which the logP value is 2.
By fixing 5 or more compounds, hydrogel particles of C-cellulose are prepared.

There are various methods for adsorbing and removing chemokines from body fluids using the adsorbent of the present invention. The simplest method is to extract the body fluid, store it in a bag or the like, mix it with an adsorbent to adsorb and remove the chemokine, and then filter the adsorbent to obtain the body fluid from which the chemokine has been removed. In addition, there is a method in which a container equipped with a filter having a body fluid inlet and an outlet, through which the body fluid passes but the adsorbent does not pass, is filled with the adsorbent, and the body fluid is allowed to flow into the container. Either method can be used, but the latter method is easy to operate, and by incorporating it into the extracorporeal circulation circuit, it is possible to efficiently remove chemokines online from the body fluid of the patient, especially blood. The adsorbent of the invention is suitable for this method.

In the extracorporeal circulation circuit here, the adsorbent of the present invention can be used alone, but it can also be used in combination with other extracorporeal circulation treatment systems. Examples of the combined use include an artificial dialysis circuit and the like, which can also be used in combination with dialysis therapy.

Next, a chemokine adsorber of the present invention using the above chemokine adsorbent will be described with reference to FIG. 1, which is a schematic sectional view of an embodiment.

In FIG. 1, 1 is a body fluid inflow port, 2 is a body fluid outflow port, 3 is a chemokine adsorbent of the present invention, and 4 and 5 are bodily fluids and components contained in the body fluid, but the chemokine adsorbents Is a filter that cannot pass, 6 is a column,
7 is a chemokine adsorber. However, the chemokine adsorber is not limited to such a specific example, and has a liquid inlet and an outlet, and a container equipped with a chemokine adsorbent outflow prevention device, the adsorbent Any material may be used as long as it is filled with.

The outflow prevention tool includes mesh, non-woven fabric,
Examples include filters such as cotton plugs. The shape, material, and size of the container are not particularly limited, but preferred specific examples include, for example, a capacity of about 150 to 500 ml,
Examples thereof include transparent or semitransparent cylindrical containers having a diameter of about 4 to 10 cm. The material is particularly preferably a material having sterilization resistance, and specific examples thereof include silicon-coated glass, polypropylene, polyvinyl chloride, polycarbonate, polysulfone, and polymethylpentene.

[0074]

EXAMPLES The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to the following Examples. Further, in the following examples, the chemokine to be adsorbed is M, which is one of CC subfamilies.
Although IP-1α is taken as an example, it goes without saying that other chemokines can be similarly implemented.

Reference Example A glass column (both inner diameter 9 mm, column length 150 mm) equipped with a filter having a pore size of 15 μm on both ends was placed on an agarose gel (Bio-Rad A-5 m manufactured by Bio-rad, particle size). 50-100 mesh), vinyl polymer gel (Toyopearl HW-65 manufactured by Tosoh Corporation, particle size 50-100 μm) and cellulose gel (Cellulofine GC- manufactured by Chisso Corporation).
(700 m, particle size 45 to 105 μm) were uniformly filled, and water was flown by a peristaltic pump to determine the relationship between the flow velocity and the pressure loss ΔP. The result is shown in FIG.

As shown in FIG. 2, Toyopearl HW-65
The flow velocity of Cellulofine GC-700m increases almost in proportion to the increase of pressure, while that of Biogel (Bi
It can be seen that the ogel) A-5m causes consolidation and that the flow velocity does not increase even if the pressure is increased. In the present invention, as in the former case, the relationship between the pressure loss ΔP and the flow velocity is 0.3.
What has a linear relationship up to kg / cm ² is called a hard gel.

Example 1 Cellulofine GC-7 which is a cellulosic porous carrier
Water was added to 170 ml of 00m (molecular weight exclusion limit of globular protein of 400,000 manufactured by Chisso Co., Ltd.), and the total amount was 340m.
After the volume was set to 1, 90 ml of a 2M sodium hydroxide aqueous solution was added and the temperature was raised to 40 ° C. 31 ml of epichlorohydrin
Was added and reacted at 40 ° C. for 2 hours with stirring. After completion of the reaction, it was thoroughly washed with water to obtain an epoxidized carrier.

To 10 ml of this epoxidized carrier was added 200 mg of n-octylamine (logP = 2.90), and 5
In a 0 (v / v)% aqueous ethanol solution at 45 ° C.,
The reaction was carried out for 6 days. After completion of the reaction, the n-octylamine-immobilized carrier was sufficiently washed in the order of 50 (v / v)% ethanol aqueous solution, ethanol, 50 (v / v)% ethanol aqueous solution, and water. The fixed amount of n-octylamine was 184 μmol per dry weight (1 g) of the carrier.

This immobilized carrier and Cellulofine GC
Human gene recombinant MIP-1α was added to normal human serum (manufactured by Dainippon Pharmaceutical Co., Ltd.) for 0.5 ml each of −700 m
(R & D systems (R & D sy
3) MIP-1α supplemented healthy human serum (MIP-1α concentration 1.1 ng / ml) prepared by adding
Was added and adsorbed under shaking at 37 ° C. for 2 hours. The concentration of MIP-1α in the supernatant solution before and after adsorption is
The measurement was performed using a human MIP-1α measurement kit manufactured by D Systems Inc., and the adsorption rate was calculated by the following formula.

[0080]

[Equation 1]

However, the concentration in the pre-adsorption serum in this case is a value obtained by previously correcting the water content in the carrier.

The albumin concentration before and after adsorption was measured by the BCG method.

Results <Adsorption rate of MIP-1α> Adsorption rate (%) Cellulofine GC-700m 0 n-octylamine-immobilized GC-700m 58 <Change in albumin concentration (unit: g / dl)> Adsorption before adsorption After Cellulofine GC-700m 3.8 3.6 n-octylamine-immobilized GC-700m 3.8 3.3

Example 2 n-octylamine was converted to cetylamine (Σf = 7.22)
A cetylamine-immobilized carrier was obtained in the same manner as in Example 1, except that the solvent for the immobilization reaction was changed to ethanol. The fixed amount of cetylamine is 1 per dry weight (1 g) of the carrier.
It was 89 μmol. Using this immobilized carrier, an adsorption experiment was conducted in the same manner as in Example 1, the adsorption rate was calculated by measuring the concentration of MIP-1α, and the change in albumin concentration was also measured.

Results <Adsorption rate of MIP-1α> Adsorption rate (%) Cetylamine-immobilized GC-700m 99 <Change in albumin concentration (unit: g / dl)> Before adsorption After adsorption Cetylamine-immobilized GC-700m 3. 8 3.3

Example 3 Cellulofine GC-700m was replaced with Cellulofine GC-
An n-octylamine-immobilized carrier was obtained in the same manner as in Example 1 except that the amount was changed to 200 m (Chisso Corporation, molecular weight exclusion limit of spherical protein: 140,000). The fixed amount of n-octylamine was 18 per dry weight (1 g) of the carrier.
It was 9 μmol. Example 1 using this immobilized carrier
An adsorption experiment was carried out in the same manner as above, the adsorption rate was calculated by measuring the concentration of MIP-1α, and the change in albumin concentration was also measured.

Result <Adsorption rate of MIP-1α> Adsorption rate (%) Cellulofine GC-200m 0 n-octylamine-immobilized GC-200m 59 <Change in albumin concentration (unit: g / dl)> Adsorption before adsorption Post-n-octylamine-immobilized GC-200m 3.8 3.7

Example 4 Cellulofine GC-700m was replaced with Cellulofine GC-
A cetylamine-immobilized carrier was obtained in the same manner as in Example 1 except that n-octylamine was changed to cetylamine and the solvent for the immobilization reaction was changed to ethanol at 200 m. The fixed amount of cetylamine is 189μ per dry weight (1g) of the carrier.
It was mol. Using this immobilized carrier, an adsorption experiment was conducted in the same manner as in Example 1, the adsorption rate was calculated by measuring the concentration of MIP-1α, and the change in albumin concentration was also measured.

Results <Adsorption rate of MIP-1α> Adsorption rate (%) Cetylamine-immobilized GC-200m 99 <Change in albumin concentration (unit: g / dl)> Before adsorption After adsorption Cetylamine-immobilized GC-200m 3. 8 3.7

Example 5 Commercially available cellulose acetate was dissolved in a mixed solvent of dimethyl sulfoxide and propylene glycol, and this solution was subjected to the method (vibration method) described in JP-A-63-117039.
Spherical hydrogel particles of cellulose acetate were obtained by making the solution into droplets and solidifying. Hydrolysis reaction is carried out by mixing these particles with an aqueous solution of sodium hydroxide to give cellulose hydrogel particles (the exclusion limit molecular weight of spherical proteins is 3
It was 0000. (Hereinafter referred to as C-1).

Cellulofine GC-700m is replaced with C-1
Then, a cetylamine-immobilized carrier was obtained in the same manner as in Example 1 except that n-octylamine was changed to cetylamine and the solvent for the immobilization reaction was changed to ethanol. The fixed amount of cetylamine was 160 μmol per dry weight (1 g) of the carrier. Using this immobilized carrier, an adsorption experiment was conducted in the same manner as in Example 1, the adsorption rate was calculated by measuring the concentration of MIP-1α, and the change in albumin concentration was also measured.

Result <Adsorption rate of MIP-1α> Adsorption rate (%) C-10 Cetylamine-immobilized C-199 <Change in albumin concentration (unit: g / dl)> Before adsorption Cetylamine-immobilized C after adsorption -1 3.8 3.6

Comparative Example 1 n-octylamine was replaced with n-butylamine (logP =
An n-butylamine-immobilized carrier was obtained in the same manner as in Example 1 except that the carrier was changed to 0.97). Using this immobilized carrier, an adsorption experiment was conducted in the same manner as in Example 1, and MIP-1α
The adsorption rate was calculated by measuring the concentration of A, and the change in albumin concentration was also measured.

Results <Adsorption rate of MIP-1α> Adsorption rate (%) n-butylamine-immobilized GC-700 m 1 <Change in albumin concentration (unit: g / dl)> Before adsorption After-adsorption n-butylamine-immobilized GC -700m 3.8 3.4

Comparative Example 2 n-octylamine was replaced with n-hexylamine (logP =
2.06) except that n-
A hexylamine-immobilized carrier was obtained. Using this immobilized carrier, an adsorption experiment was conducted in the same manner as in Example 1, and MIP-
The adsorption rate was calculated by measuring the concentration of 1α, and the change in albumin concentration was also measured.

Results <Adsorption rate of MIP-1α> Adsorption rate (%) n-hexylamine-immobilized GC-700m 6 <Change in albumin concentration (unit: g / dl)> Before adsorption, after adsorption n-hexylamine fixation GC-700m 3.8 3.3

[0097]

The water-insoluble carrier according to the method of the present invention is
By using an adsorbent characterized by immobilizing a compound having a gP value of 2.50 or more, chemokines in body fluid can be efficiently adsorbed and removed.

[Brief description of drawings]

FIG. 1 is a schematic sectional view of an embodiment of a chemokine adsorber of the present invention.

FIG. 2 is a graph showing the results of investigating the relationship between flow velocity and pressure loss using three types of gels.

[Explanation of symbols]

 1 Body fluid inlet 2 Body fluid outlet 3 Chemokine adsorbent 4, 5 Filter 6 Column 7 Chemokine adsorber

Claims (6)

[Claims] 1. A chemokine in a body fluid (however, interleukin-8 is characterized in that a compound having a log P (P is a partition coefficient in an octanol-water system) value of 2.50 or more is immobilized on a water-insoluble carrier. (Excluding) adsorbent. 2. An adsorbent for a chemokine (excluding interleukin-8) in a body fluid according to claim 1, wherein the water-insoluble carrier is a hydrophilic carrier. 3. An adsorbent for a chemokine (excluding interleukin-8) in a body fluid according to claim 1 or 2, wherein the water-insoluble carrier has a porous structure. 4. Adsorption of a chemokine (excluding interleukin-8) in a body fluid according to claim 1, 2 or 3, wherein the exclusion limit molecular weight of the globular protein of the water-insoluble carrier is 10,000 or more and 600,000 or less. Material. 5. A chemokine (excluding interleukin-8) in a body fluid, which comprises contacting a body fluid with an adsorbent of the chemokine (excluding interleukin-8) according to claim 1. Adsorption removal method. 6. A container having an inlet and an outlet for liquid and having means for preventing the adsorbent from flowing out of the container.
An adsorber for chemokines (excluding interleukin-8) in body fluids, which is filled with an adsorbent of the described chemokines (excluding interleukin-8).