JP5298719B2 - Leukocyte removal column - Google Patents

Description

  The present invention relates to a leukocyte removal column.

  Inflammatory diseases such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, Behcet's disease, systemic lupus erythematosus, adult Still's disease and acute lung injury (ARDS / ALI) Infections caused by bacteria or viruses are considered as one of the causes.

  In inflammatory diseases, the inflammatory response is repeatedly enhanced, and excessive granulocytes, monocytes, macrophages or dendritic cells are induced in the exudate or tissue around the tissue where blood and inflammatory responses are observed. When granulocytes recognize foreign antigens or self-antigens, they mutually interact with monocytes, macrophages, dendritic cells, etc., and as a result, induced chemokines such as interleukin-8 act in inflammatory tissues. Be guided to. In addition, granulocytes are known to cause tissue damage at an accelerated rate when activated by the progression of an inflammatory reaction.

  Furthermore, interleukin-1β and TNF-α activate neutrophils among granulocytes, and the activated neutrophils phagocytose antigens and cause degranulation, elastase having tissue damage, By producing nitric oxide, hydrogen peroxide, etc., inflammation and damage of the tissue will progress. Therefore, in order to effectively treat inflammatory diseases, it is important to remove excess granulocytes and activated granulocytes only when the inflammatory reaction is progressing.

  Conventionally, many blood processing columns using granulocytes as an adsorption removal target have been developed (Patent Documents 1 to 3), and adsorption carriers that selectively remove activated granulocytes have been reported (Patent Documents). 4 and 5).

  However, there is no example in which the therapeutic effect or biocompatibility of inflammatory diseases is examined based on the filling rate of the adsorbent carrier and the removal rate ratio between non-inflammatory and inflammatory conditions. Here, “biocompatibility” refers to a property that does not impair the immune function of the living body, but as excellent biocompatibility, for example, granulocytes that are necessary for infection protection during non-inflammation are unnecessary. For example, a characteristic that does not remove many lymphocytes unnecessarily so as not to become easily infectious.

  Adsorption carriers that selectively remove granulocytes and monocytes using cellulose acetate beads as an adsorbent carrier material have been developed. Although the mechanism by which this adsorption carrier removes granulocytes and monocytes is still unclear, it is speculated that the activated complement component plays an important role in activating granulocytes. Yes. (Non-Patent Document 1).

  However, the ability to adsorb and remove granulocytes of the above-mentioned adsorption carrier is affected by the degree of complement activation, and since activated granulocytes are created and removed even during non-inflammation, before and after inflammation. There is no difference in the removal rate, and unnecessarily many granulocytes are removed. Thus, utilization of cellulose acetate as a material for the adsorption carrier is concerned from the viewpoint of biocompatibility.

  Furthermore, a material for removing white blood cells has been developed by using a filter using a polyester nonwoven fabric, regardless of the type of white blood cells.

However, the above-mentioned filter has the same ability to remove leukocytes between non-inflammation and inflammation, and about 90% or more of leukocytes are removed even in non-inflammation. For this reason, granulocytes at the time of non-inflammation necessary for foreign body recognition or lymphocytes that play an important role in the immune mechanism are also removed, so there is still concern from the aspect of biocompatibility (Patent Document 6). .
JP-A-60-193468 JP-A-5-168706 JP 2002-172163 A JP 1999-244663 A JP 2002-539178 A JP-A-8-103493 Suzuki et al., Japanese Society of Apheresis, 2001, Volume 20, No. 1, p. 17-26

  Accordingly, an object of the present invention is to provide granules related to tissue injury that increases in body fluids during inflammation without removing unnecessarily many granulocytes and monocytes necessary for defense against infection present in body fluids during non-inflammation. An object of the present invention is to provide a leukocyte removal column that can efficiently and selectively remove spheres and monocytes and has both excellent therapeutic effect on inflammatory diseases and biocompatibility.

  In order to achieve the above problems, the present inventors have conducted extensive research and can efficiently remove granulocytes that increase in body fluids during inflammation, compared with granulocytes present in body fluids during non-inflammation. The present inventors have found a leukocyte removal column that can selectively remove granulocytes present in body fluids during inflammation.

That is, the present invention provides the leukocyte removal column described in the following (1) to (6).
(1) A leukocyte removal column for efficiently removing granulocytes from a body fluid, wherein the adsorption carrier filled in the leukocyte removal column is a fiber having an average diameter of 0.5 to 30 μm or less, and the adsorption carrier is filled The rate is 9.8 to 19.5% by volume, and the removal rate of granulocytes from the body fluid at the time of inflammation is 1.2 times or more compared with the removal rate of granulocytes from the body fluid at the time of non-inflammation. There is a leukocyte removal column.
(2) The leukocyte removal column according to (1), wherein the fiber has a functional group having an amino group on the surface.
(3) The leukocyte removal column according to (1) or (2), wherein the adsorption carrier composed of the fibers has a sheet structure.
(4) removing at least one cytokine from the group consisting of interleukin-1β, interleukin-6, interleukin-8, interleukin-10, TNF-α, (1) to (3) The leukocyte removal column according to any one of the above.
(5) The leukocyte removal column according to any one of (1) to (4), which is used for treatment of an inflammatory disease.
(6) The leukocyte removal column according to (5), wherein the inflammatory disease is rheumatoid arthritis.

  According to the leukocyte removal column of the present invention, granulocytes that increase in the body fluid at the time of inflammation can be efficiently and selectively removed, and effective treatment for inflammatory diseases and excellent biocompatibility can be achieved. realizable.

  Hereinafter, preferred embodiments for carrying out the present invention will be described.

  The leukocyte removal column of the present invention is a leukocyte removal column that efficiently removes granulocytes from a body fluid, and the adsorption carrier packed in the leukocyte removal column is a fiber having an average diameter of 0.5 to 30 μm or less, The filling rate of the adsorption carrier is 9.8 to 19.5% by volume, and the removal rate of granulocytes from the body fluid at the time of inflammation is 1 compared with the removal rate of granulocytes from the body fluid at the time of non-inflammation. . It is characterized by being twice or more.

  “Body fluid” refers to a liquid that exists in the body of humans and animals, and specifically, a liquid or a biological tissue that fills a space between tissues, a body cavity, or a tube or circulatory system that spreads throughout the body. Or the liquid which exudes from a cell. For example, blood, lymph, tissue fluid, ascites, pleural effusion, amniotic fluid or joint fluid can be mentioned.

  “Granulocytes” are blood cells characterized by occupying 60% or more of leukocytes and having granules in the cytoplasm, and are classified as neutrophils, eosinophils and basophils.

  “At the time of inflammation” refers to a state in which human and animal body tissues are undergoing an inflammatory reaction, and “at the time of non-inflammation” refers to a state other than at the time of inflammation. In clinical findings, perception disorders such as pain and movement disorders are also included during inflammation.

  Here, the “inflammatory reaction” refers to a protective reaction that occurs when a living body receives some harmful stimulus. Examples of harmful stimuli include pathogens such as bacteria and viruses, self-antigens, and physical substances such as heat and radiation. Examples include stimuli, chemical stimuli such as acids, alkalis and drugs, and other allergic reactions. Symptoms caused by an inflammatory reaction include local redness, fever, swelling, bleeding, ulcers, and pain. In vivo, living tissues that have received harmful stimuli, exudates from the living tissues or cells, or It is characterized by an increase or activation of granulocytes in the blood.

The activation of granulocytes is determined by the expression of genes such as cytokines and chemokines in granulocytes and the high expression of these translation products, such as neutrophils and eosinophils. Or the state in which the base sphere is in the following state can be exemplified.
1) When a neutrophil recognizes a foreign substance that is a harmful stimulus, the neutrophil takes the foreign substance into the cell and digests, sterilizes, or decomposes the foreign substance with an enzyme (myeloperoxidase, elastase, muramitase, etc.) present in the intracellular vesicle. However, these enzymes are also released extracellularly, increase vascular permeability, lead to neutrophil recruitment to tissues that have recognized foreign bodies, and cause an inflammatory reaction. As a result, neutrophils interact with monocytes, macrophages, dendritic cells, etc. in tissues that have undergone an inflammatory reaction and are activated, releasing cytokines, chemokines, active oxygen, etc. Will be shown. Thus, the state in which neutrophils infiltrate the inflamed tissue and exhibits tissue damage corresponds to the activation of the granulocytes and is included during inflammation.
2) When eosinophils recognize foreign substances that are harmful stimuli, like neutrophils, they show motility, chemotaxis, and phagocytosis, and the number of cells increases. , Releases proteins called eosinophil compressing protein, eosinophil derived neurotoxin and eosinophil peroxidase, and exhibits tissue damage. Thus, the state in which eosinophils increase and shows tissue damage corresponds to the activation of granulocytes, and is included during inflammation.
3) Basophils are known to play an important role in the progression of allergic inflammatory reactions. When foreign substances that cause harmful stimuli are recognized, they degranulate and become histamine, platelet-activating factor, serotonin. And heparin are released, and the permeability of blood vessels is increased, causing airway contraction observed in sneezing, rhinitis, and asthma. Thus, the state where basophils are degranulated corresponds to the activation of granulocytes described above and is included during inflammation.

  The adsorption carrier is a fiber, and examples of the fiber material include an organic polymer compound, an inorganic polymer compound, a metal, and the like, and an organic polymer compound is more preferable. Examples of the organic polymer compound include polyamides such as polyacrylonitrile, polystyrene, nylon (registered trademark) 6, nylon (registered trademark) 66, polyolefin, polyester, and the like.

  Polyolefin refers to a polymer obtained by homopolymerization or copolymerization of alkenes or alkynes. For example, polyolefins obtained by homopolymerization of polyethylene, polypropylene, polybutylene, etc., or polypropylene-polyethylene copolymers, polybutylene-polypropylene copolymers. Examples thereof include polyolefins obtained by copolymerization of polymers and the like.

  Examples of the polyester include polyethylene terephthalate, polybutylene terephthalate, and polyoxyethylene terephthalate. In order to introduce a functional group capable of adsorbing cytokines into the adsorption carrier, in particular, polystyrene or polysulfone having an aromatic ring is more preferable.

  In order to remove granulocytes using the phagocytosis of granulocytes, the average diameter of the fibers serving as the adsorption carrier is preferably 0.5 to 30 μm or less, and more preferably 4 to 10 μm. Preferably, it is 5-10 micrometers. Further, the cross-sectional shape of the fiber is preferably a circular cross-section, but any other irregular cross-section can be applied.

  The average diameter of the adsorption carrier is a value obtained by randomly collecting 10 samples of the adsorption carrier, taking a photograph of 1000 to 3000 times with a scanning electron microscope, and measuring the diameter of 10 places (100 places in total) per sample. It means the average value of. When the measurement cross section of the adsorption carrier sample is an ellipse, rectangle, or polygon, the diameter of the circle corresponding to the area of the figure formed by connecting the outermost layers is the uneven shape where the protrusions exist. The diameter of the circle corresponding to the area of the figure formed by connecting the vertices of all the protrusions may be adopted as data for calculating the average diameter.

  The fiber preferably has a functional group having an amino group on the surface, and the ability to adsorb cytokine can be further enhanced by the amino group.

  Examples of the functional group having an amino group include a cyclic peptide residue having an amino group, a polyalkyleneimine residue, a benzylamino group, or a primary to tertiary alkylamino group. An amino group or a polyalkyleneimine residue is preferable, and a primary to tertiary alkylamino group is more preferable.

  The cyclic peptide having an amino group may be any peptide having one or more amino groups in the side chain, but is preferably a cyclic peptide consisting of 2 to 50 amino acids, and consisting of 4 to 16 amino acids. Cyclic peptides are more preferred. For example, suitable specific examples include polymyxin B, polymyxin E, colistin, gramicidin S, and alkyl derivatives and acyl derivatives thereof.

  Examples of the polyalkyleneimine residue include polyethyleneimine, polyhexamethyleneimine, polyalkyleneimine typified by a poly (ethyleneimine / decamethyleneimine) copolymer, and some nitrogen atoms of the polyalkyleneimine are halogenated. Alkylated hydrocarbons (for example, n-hexyl bromide, n-decanyl bromide, n-stearyl bromide, etc.) or mixtures thereof, or a part of the nitrogen atom of the polyalkyleneimine is butyric acid, valeric acid, lauryl And acylated with a fatty acid such as acid, myristic acid, lenolenic acid and stearyl acid.

  As the amino group, a primary to tertiary amino group is preferable, and from the viewpoint of adsorbability to cytokines, a primary to tertiary amino group having 3 to 18 carbon atoms per nitrogen atom is more preferable. A primary to tertiary amino group having 4 to 18 carbon atoms per unit is more preferable.

  Specific examples thereof include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl-hexylamine, tetraethylenepentamine and the like. However, tetraethylenepentamine is particularly preferable in order to increase the ability to adsorb cytokines described below. In addition, when a plurality of amino groups are present in the functional group having the amino group, each amino group must be present on the same chain of the functional group as a functional group derived from tetraethylenepentamine. Is preferred.

  The density of amino group bonds is preferably 0.01 to 2.0 mol or less, more preferably 0.1 to 1.0 mol or less, per repeating unit of the adsorption carrier. The density of amino group bonds varies depending on the chemical structure and application of the adsorption carrier, but if it is too small, its function tends not to be expressed, while if too large, the physical strength of the adsorption carrier after immobilization is low. It tends to be worse and the function as an adsorption carrier tends to be lowered.

  The adsorption carrier composed of the above fibers is preferably sheet-like, and examples of the sheet-like fibers include felt, knitted fabric, woven fabric, and net. The sheet-like fibers can be produced by using known techniques available to those skilled in the art. For example, a known nonwoven fabric production method such as a carding method, an air lay method, a spun bond method, or a melt blow method can be used.

  In particular, when the sheet-like adsorption carrier is a non-woven fabric, it is preferable to have a structure of two or more layers with a net in order to improve the shape retention. Such a structure of two or more layers mainly indicates a laminated structure. The laminated structure may be a two-layer structure of a nonwoven fabric and a net, but it is more preferable to take a shape in which a net is sandwiched between nonwoven fabrics, that is, a nonwoven fabric-net-nonwoven fabric sandwich structure.

  The leukocyte removal column is preferably capable of removing one or more cytokines that are indicated to be involved in ulcerative colitis, Crohn's disease, rheumatoid arthritis, acute lung injury, and the like. As such cytokines, interleukin- Examples include 1β, interleukin-6, interleukin-8, interleukin-10, and TNF-α.

  The leukocyte removal column can be prepared by aseptically filling the adsorption carrier into the column by a method commonly used by those skilled in the art, and the packing rate is 9.8 to 19.5% by volume. It is preferable that it is 12.7 to 18.5 volume%.

  When the filling rate of the adsorption carrier is 7.8% by volume or less, the removal rate of granulocytes at the time of inflammation is 35% or less, and it is predicted that a sufficient effect for treating inflammatory diseases cannot be obtained. . On the other hand, when the filling rate is 21.5% by volume or more, the removal rate of lymphocytes that are not preferably removed in the treatment of inflammatory diseases is significantly increased, and the immune function of patients with inflammatory diseases is reduced, Furthermore, the therapeutic effect may be reduced.

  Here, the filling rate is a value obtained by dividing the volume of the adsorption carrier by the volume of the adsorption carrier filling portion and multiplying by 100.

Below, the measuring method of the volume of an adsorption carrier and the volume of an adsorption carrier filling part is prescribed | regulated.
(Measurement method of volume of adsorption carrier)
The specific gravity of ultrapure water is assumed to be 1 and converted to volume.

  First, prepare a graduated cylinder that contains the adsorption carrier, put the adsorption carrier taken out of the column into it, and fill the ultrapure water with no gap until the adsorption carrier is sufficiently submerged. Measure and obtain the value (mL) as volume A. At that time, care should be taken not to leave bubbles.

  Thereafter, the adsorption carrier is taken out from the measuring cylinder, the wet mass of the adsorption carrier containing water is measured with an electronic balance, and the obtained value (g) is defined as a wet mass B. At this time, care should be taken not to spill water contained in the adsorption carrier. The adsorbent carrier is dried with a vacuum dryer set at 30 ° C., the dry mass of the adsorbent carrier is measured with an electronic balance, and the obtained value (g) is defined as dry mass C. In order to judge that it has dried, it can be used as an indicator that the mass difference when the dry mass is measured twice is 1% or less. The interval for measuring the dry mass twice is 1 hour.

  Finally, the volume of ultrapure water remaining after the adsorption carrier is taken out is measured with a graduated cylinder, and the obtained value is (mL) as volume D.

  As a result, the volume of the adsorption carrier is calculated by the following formula 1.

Volume of adsorption carrier = volume A − ((wet mass B−dry mass C) + volume D).
(Measurement method of the volume of the adsorption carrier packed part)
The volume of the adsorption carrier filling portion means the volume of the portion filled with the adsorption carrier, and does not include other members for defining the blood flow path or the space of the flow path itself. For example, if there is a gap for the blood flow path between the adsorption carrier and the cylindrical tube, the calculation is performed. The outermost layer of the adsorption carrier often has fine irregularities, but is considered as a surface. It can be easily determined by measuring and calculating using calipers. Even when the shape is complicated and measurement is impossible, it can be easily obtained by actually measuring each member.

  In the leukocyte removal column, the removal rate of granulocytes from the body fluid at the time of inflammation is preferably 1.2 times or more compared to the removal rate of granulocytes from the body fluid at the time of non-inflammation. More preferably, it is more than twice, and more preferably 1.7 times or more. In this case, from the viewpoint of maintaining the immune function, the leukocyte removal column preferably has a lymphocyte removal rate of 50% or less, and more preferably 30% or less.

  As already explained, granulocytes during inflammation are activated, and activated granulocytes can cause inflammatory diseases, while non-activated granulocytes are important in maintaining the immune function of healthy people Therefore, the ability to selectively remove granulocytes at the time of inflammation more than 1.2 times is very useful for exerting therapeutic effects without lowering the immune function of patients with inflammatory diseases. is there. In addition, the removal rate of granulocytes is calculated by the following formula 2.

Granulocyte removal rate (%) = {(number of granulocytes at the column inlet−number of granulocytes at the column outlet) / number of granulocytes at the column inlet} × 100 ··· formula 2
“The number of granulocytes at the column inlet” and “the number of granulocytes at the column outlet” in the formula can be easily measured by using, for example, an automatic blood cell counter. Examples of the automatic blood cell measuring device include XT-1800i (Sysmex Corporation).

  Moreover, the value which compared the removal rate of the granulocyte from the bodily fluid at the time of inflammation and the removal rate of the granulocyte from the bodily fluid at the time of non-inflammation is calculated by the following formula | equation 3 as a removal rate ratio.

Removal rate ratio = removal rate of granulocytes from body fluids during inflammation / removal rate of granulocytes from body fluids during non-inflammation ··· Equation 3
The higher the removal rate ratio, the better. The leukocyte removal column that has been used in the technical field of leukocyte removal columns has a function of removing leukocytes at a higher rate during inflammation than in non-inflammation. Is not known. When the removal rate ratio is 1.2 or more, the removal of leukocytes necessary for infection protection existing at the time of non-inflammation is suppressed, and leukocytes that can be expected to have a more therapeutic effect on patients with inflammatory diseases are selectively removed. Can be expected to show a therapeutic effect.

As an evaluation method of the granulocyte removal rate ratio, the following method was used.
(Evaluation method of granulocyte removal rate ratio)
1) Preparation of a rabbit arthritis model A mixed solution in which ovalbumin (Sigma) and Freund's complete adjuvant were mixed at a mass ratio of 1: 1 (4 mg each) was added to a rabbit (Japanese white species, male, approximately weight). 3 kg) and 14 days after the administration, a mixed solution of ovalbumin and Freund's complete adjuvant having the same composition as above is prepared again, and the second administration is performed. Five days after the second administration, ovalbumin is administered to the right knee joint of the same rabbit to induce arthritis, and a rabbit arthritis model is prepared. The left knee joint is administered with physiological saline instead of ovalbumin as a control. In this evaluation, 3 or more rabbits are used.
2) Evaluation of granulocyte removal rate in the rabbit arthritis model For the rabbit arthritis model prepared by the above method, the extracorporeal circulation of blood is performed using a column packed with an adsorption carrier to evaluate the leukocyte removal performance . The amount of heparin used for extracorporeal circulation is 30 IU / kg / hr (heparin-added physiological saline concentration: 20 IU / mL). The extracorporeal circulation of blood is performed using the rabbit ear arteriovenous at 10 minutes (no inflammation) and 24 hours (inflammation) after challenge. The blood flow rate was 2 mL / min for 30 minutes each, and when 30 minutes had passed after the end of extracorporeal circulation, blood was collected before and after the entrance of the column, and the number of granulocytes (if necessary) The number of lymphocytes in addition to granulocytes is measured with an automatic blood cell counter. The number of granulocytes in the blood collected from the inlet of the column (the number of granulocytes at the inlet of the column) and the number of granulocytes in the blood collected from the outlet (the granulocytes at the outlet of the column) The removal rate of granulocytes is calculated by applying the number) to Equation 2 above. The removal rate is evaluated using an average value of values obtained with three or more birds.
3) Removal rate ratio The removal rate ratio is obtained using the average value of the values carried out by three or more birds according to Equation 3.

  The leukocyte removal column is preferably used for the treatment of inflammatory diseases. Inflammatory diseases include, for example, ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriasis, Behcet's disease, systemic lupus erythematosus, adult Still's disease, hemophagocytic syndrome, multiple sclerosis, acute lung injury (ARDS / ALI) Rheumatoid arthritis is more suitable as an inflammatory disease in which the leukocyte removal column exhibits a therapeutic effect, such as idiopathic pulmonary fibrosis and interstitial pneumonia.

  The present invention will be described more specifically in the following examples, but the content of the invention is not limited thereto.

A rabbit arthritis model was prepared as a rheumatoid arthritis model, and the removal effect of granulocytes and lymphocytes was examined using a leukocyte removal column. The above-described methods were used to prepare a rabbit arthritis model and to measure granulocyte removal rate ratio and lymphocyte count. The collected granulocytes and lymphocytes were measured using an automatic blood cell counter (XT-1800i; Sysmex Corporation).
(Evaluation method of lymphocyte removal rate in rabbit arthritis model)
An expression for calculating an evaluation method of the lymphocyte removal rate in the rabbit arthritis model is shown below.

Lymphocyte removal rate (%) = {(number of lymphocytes at the column inlet−number of lymphocytes at the column outlet) / number of lymphocytes at the column inlet} × 100
(Evaluation of arthritis in rabbit arthritis model)
The arthritis symptoms in the rabbit arthritis model were evaluated by measuring the diameter of joint swelling using the swelling of the knee joint caused by arthritis as an index.

  Specifically, before and after 48 hours from the induction, the knee joint part of the rabbit arthritis model is bent at a right angle with a splint, and the lateral width of the knee joint part is a digital caliper (Digimatic Caliper, CD-15CX; Mitutoyo Corporation). The joint swelling diameter was calculated from the following formula, and the average value was used.

Joint swelling diameter (mm) = joint diameter A−joint diameter B
Joint diameter A (mm) = Right knee joint diameter 48 hours after initiation-Left knee joint diameter 48 hours after initiation Joint diameter B (mm) = Right knee joint diameter before induction-Left knee before induction Further, in order to confirm the presence or absence of an inflammatory reaction, the serum CRP concentration was measured before each extracorporeal circulation.

Specifically, the CRP concentration was measured by using the ELISA kit (Levis CRP-rabbit; Shibayagi Co., Ltd.) according to the method indicated in the kit.
(Production of material A)
A 36 island sea-island composite fiber, in which the island is further formed of a core-sheath composite, was obtained using the following components under spinning conditions of a spinning speed of 800 m / min and a draw ratio of 3 times.

Island core component: Polypropylene Island sheath component: Polystyrene 95% by mass, Polypropylene 5% by mass
Sea component: Copolymer polyester containing ethylene terephthalate unit as the main repeating unit and 3% by mass of 5-sodiumsulfoisophthalic acid as a copolymer component Composite ratio (mass ratio): Core: Sheath: Sea = 45: 40: 15
After producing a nonwoven fabric composed of 85% by mass of this fiber and 15% by mass of polypropylene fiber having a diameter of 20 μm, a sheet-like polypropylene net (thickness: 0.5 mm, single yarn diameter: 0.3 mm) between the two nonwoven fabrics. , Opening: 2 mm square), and needle punching was performed to obtain a carrier having a three-layer structure.

Next, this non-woven fabric is treated at 95 ° C. with a 3% by mass aqueous sodium hydroxide solution to make the ethylene terephthalate unit of the sea component the main repeating unit, and a copolymerized polyester containing 3% by mass of 5-sodium sulfoisophthalic acid as a copolymerization component. Was dissolved into a nonwoven fabric having a core-sheath fiber diameter of 4.5 μm and a bulk density of 0.025 g / cm ³ .

  Next, 46% by mass of nitrobenzene, 46% by mass of sulfuric acid, 0.8% by mass of paraformaldehyde, and 7.2% by mass of N-methylol-α-chloroacetamide (hereinafter, NMCA) were mixed, stirred and dissolved at 10 ° C. or less. , An NMCA reaction solution was prepared. The NMCA reaction solution was brought to 15 ° C., and the NMCA reaction solution was added at a solid-liquid ratio of about 40 mL to 1 g of the nonwoven fabric, and reacted for 2 hours while maintaining the reaction solution at 15 to 20 ° C. in a water bath.

Thereafter, the nonwoven fabric was taken out from the reaction solution, and immersed in nitrobenzene in the same amount as the NMCA reaction solution and washed. Subsequently, the nonwoven fabric was taken out, immersed in methanol and washed to obtain an α-chloroacetamidomethylated polystyrene nonwoven fabric (Intermediate 1). Further, 10 g of Intermediate 1 was immersed in a solution of 6.3 g of tetraethylenepentamine and 7.2 g of n-butylamine in 500 mL of dimethylformamide (DMF), reacted at 30 ° C. for 3 hours, washed with DMF, and then washed with water. Then, 13.9 g of tetraethylenepentamated fiber (hereinafter referred to as adsorption carrier A) was obtained from intermediate 1 by vacuum drying.
(Packing of adsorption carrier A into the column)
Each column is packed with adsorption carrier A punched to a diameter of 10 mm per one mini-column (diameter 10 mm × length 23.49 mm, adsorption carrier packed part volume: 1.84 cm ³ ) (packing rate: 7.8 to 21.5). Volume%). Thereafter, the minicolumn was filled with water for injection (Otsuka Pharmaceutical) and then autoclaved. The number of fillings and the filling rate are as shown in Table 1.

  The method for measuring the packing rate is the same as that described above, but here the packing rate is calculated using a mini-column. In calculating the packing rate of the mini-column, the volume of the adsorption carrier packed portion was calculated by taking into consideration the space volume not filled with the adsorption carrier as seen in the product form column, and the packing rate was obtained. The formula for calculating the filling rate (volume%) is shown below.

Filling rate (% by volume) = volume of adsorption carrier / volume of adsorption carrier filling portion × 100
Volume of adsorbent carrier packed part = column volume-space volume not filled with adsorbent carrier (volume of the part filled only with body fluid when the column is used)
(Packing of cellulose acetate beads into a column)
A cellulose acetate bead having a diameter of about 2 mm was prepared, and 13.06 g was filled in a silicon tube having a diameter of 15 mm, and fittings for connection with a circuit were attached to the top and bottom thereof (adsorption carrier volume: 10.07 cm ³ ). Thereafter, the inside of the silicon tube was filled with a physiological saline solution (Otsuka Pharmaceutical Co., Ltd.), and a column having an inner diameter of 15 mm and a length of 105 mm was produced (volume of adsorption carrier packed part: 18.6 cm ³ ). The produced column was autoclaved.
(Examples 1-4)
The extracorporeal circulation was performed according to the above-described method for evaluating the removal ratio of granulocytes, and the column filled with the adsorption carrier A was evaluated (1 group: N = 3). The filling rate of the adsorption carrier A was 9.8, 12.7, 18.5, and 19.5% by volume. The results are shown in Table 2 and FIG.
(Comparative Example 1)
The extracorporeal circulation was performed according to the above-described method for evaluating the removal ratio of granulocytes, and the column filled with the adsorption carrier A was evaluated (1 group: N = 3). The filling rate of the adsorption carrier A was 21.5% by volume. The results are shown in Table 2 and FIG.

  From the results in Table 2, when the filling rate of the adsorption carrier A is in the range of 9.8 to 19.5% by volume or less, the removal rate ratio between non-inflammation and inflammation is 1.2 or more. While the removal rate was suppressed, granulocytes during inflammation were removed at a high rate. Furthermore, the removal rate of lymphocytes was 50% or less, and it was confirmed that the biocompatibility was high also for lymphocytes (Examples 1 to 4). On the other hand, when the filling rate was 7.8% by volume, the removal rate of granulocytes at the time of inflammation was low, and the therapeutic effect using the swelling of the joint diameter as an index was low. On the other hand, when the filling rate is 21.5% by volume, the removal rate of lymphocytes responsible for immune function is increased, and the removal rate of granulocytes at the time of non-inflammation is increased. It was seen (Comparative Example 1).

From these results, by filling the adsorption carrier A with a filling rate of 9.8 to 19.5% by volume or less, the removal rate of granulocytes at the time of inflammation is 1. compared with the removal rate at the time of non-inflammation. It was shown that the leukocyte removal column had a function of removing at a high rate of 2 times or more and excellent in biocompatibility by suppressing the removal rate of lymphocytes to 50% or less.
(Comparative Example 2)
According to the method for evaluating the removal rate ratio of granulocytes, extracorporeal circulation was performed, and the column filled with cellulose acetate beads was evaluated (1 group: N = 3). The results are shown in Table 3.

From the results in Table 3, it can be seen that in the column packed with cellulose acetate beads, the removal rate of granulocytes from blood during inflammation is less than 1 compared to the removal rate of granulocytes from blood during non-inflammation. The removal rate of granulocytes during non-inflammation was slightly higher than that during inflammation. That is, it did not have a function of removing granulocytes that increase at the time of inflammation at a high rate, and the removal rate of granulocytes was similar at the time of non-inflammation and inflammation.
(Examples 5 to 8)
According to the arthritis evaluation method of the above-mentioned rabbit arthritis model, extracorporeal circulation was performed, and the removal rate of granulocytes, the removal rate ratio of granulocytes, and the joint swelling diameter were evaluated using a column filled with adsorption carrier A did. The filling rate of the adsorption carrier A was 9.8, 12.7, 18.5, and 19.5% by volume. The results are shown in Table 4.
(Comparative Example 3)
According to the arthritis evaluation method of the above-mentioned rabbit arthritis model, extracorporeal circulation was performed, and the joint swelling diameter was evaluated using a column filled with the adsorption carrier A. The filling rate of the adsorption carrier A was 7.8% by volume. The results are shown in Table 4.
(Comparative Example 4)
According to the arthritis evaluation method of the above-mentioned rabbit arthritis model, extracorporeal circulation was performed, and a Sham column not filled with an adsorbent was evaluated (1 group: N = 3). As the Sham column, a silicon tube having a diameter of 7 mm was prepared, and fittings for connection to the circuit were attached to the top and bottom to prepare a column having an inner diameter of 7 mm × a length of 100 mm (priming capacity: 4.08 mL). Thereafter, EOG sterilization was performed. The results are shown in Table 4.

  From the results of Table 4, the column packed with 9.8 to 19.5% by volume or less of the adsorbent carrier A has been found to have an effect of suppressing the joint swelling diameter as the granulocytes are removed at a high rate during inflammation. (Examples 5-8). On the other hand, a column packed with 7.8% by volume of the adsorption carrier A shows a granulocyte removal rate ratio of 1.2 or more, but the granulocyte removal rate during inflammation is low and the joint swelling diameter is also suppressed. It was shown to be low (Comparative Example 3). In the Sham column, the removal rate of granulocytes at the time of inflammation was extremely low, and the effect of suppressing the joint swelling diameter was not observed (Comparative Example 4). From the above, it was shown that the column packed with 9.8 to 19.5% by volume or less of the adsorption carrier A removes granulocytes at a high rate at the time of inflammation. Suppression of inflammation was shown, and the therapeutic effect of inflammation was shown.

  The leukocyte removal column of the present invention can effectively remove the granulocytes that increase in the body fluid during inflammation, so that the therapeutic effect of the inflammatory disease is enhanced and it is used for the treatment of the inflammatory disease having excellent biocompatibility. Can do.

It is the graph which showed the relationship between adsorption carrier filling rate (%) and granulocyte removal rate ratio.

Claims (7)

Leukocyte removal column der to efficiently remove granulocytes from body fluids it is,
Adsorption carrier to be filled in the leukocyte-removing column, the average diameter is less fiber 0.5 to 30 m, the filling rate of the adsorption carrier is 9.8 to 19.5% by volume, leukocyte removal column. The leukocyte removal column according to claim 1, wherein the removal rate of granulocytes from the body fluid at the time of inflammation is 1.2 times or more compared to the removal rate of granulocytes from the body fluid at the time of non-inflammation. The leukocyte removal column according to claim 1 or 2 , wherein the fiber has a functional group having an amino group on the surface. The leukocyte removal column according to any one of claims 1 to 3 , wherein the adsorption carrier composed of the fibers has a sheet structure. Interleukin 1 beta, interleukin-6, interleukin-8, the removal of at least one kind of cytokine interleukin-10 and cytokines that will be selected from the group consisting of TNF-alpha, claim 1-4 The leukocyte removal column according to one item. The leukocyte removal column according to any one of claims 1 to 5 , which is used for treatment of an inflammatory disease. The leukocyte removal column according to claim 6 , wherein the inflammatory disease is rheumatoid arthritis.

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