JP5017996B2 - Leukocyte and cytokine adsorber - Google Patents

Description

  The present invention relates to an adsorber capable of adsorbing or removing leukocytes and cytokines from blood. More specifically, the present invention relates to a blood processing column suitable for adsorbing or removing leukocytes and cytokines.

  As is widely known, blood contains white blood cells. There are various types of leukocytes such as granulocytes, monocytes, and lymphocytes. Of these, granulocytes and monocytes are important for infection protection. However, if they increase more than necessary, they release inflammatory cytokines, active oxygen, etc. It is harmful because it continues to damage organs such as blood vessels and kidneys. For example, it causes severe sepsis, induction of coronary artery occlusion, and other autoimmune diseases such as ulcerative colitis, Crohn's disease, or rheumatism. It is necessary to adsorb or remove granulocytes and monocytes. It is also necessary to adsorb or remove cytokines and active oxygen that increase during inflammation.

  So far, columns for removing leukocytes and removing leukocytes for granulocytes (Patent Documents 1 and 2) and columns for adsorbing cytokines (Patent Documents 3 and 4) have been developed. Yes.

  In these column carriers, the efficiency for removing leukocytes themselves or cytokines has been improved, but it remains because blood cells and all humoral factors to which blood cells respond can not be removed. Cell normalization is insufficient.

  Columns that aim to adsorb both leukocytes and toxins (Patent Documents 5 and 6) have also been developed, but it cannot remove any bioactive substances produced by cells such as cytokines. Normalization of the remaining cells becomes insufficient.

In these adsorbers, it has been pointed out that clogging occurs due to the removed blood cell components, and in order to overcome this, a column (Patent Document 7) that defines voids in the adsorbent is proposed. Has been developed.
JP-A-60-193468 JP-A-5-168706 Japanese Patent Laid-Open No. 10-225515 JP-A-12-237585 Japanese Patent Laid-Open No. 14-113097 JP-A-14-172163 Japanese Patent Laid-Open No. 7-80062

  As described above, various cytokines such as cancer necrosis factor, interleukin 1, interleukin 6, and interferon, and acid peroxide are released from leukocytes, particularly excessive granulocytes and monocytes. On the other hand, it is known that these excessive cytokines have a physiological adverse effect. Therefore, the present inventors consider that it is important to improve the blood state by adsorbing or removing leukocytes that release cytokines, particularly granulocytes and monocytes, together with preventing an increase in blood levels of cytokines. It was. The object of the present invention is to efficiently adsorb or remove leukocytes typified by granulocytes or monocytes from the blood by adhering them to the water-insoluble material incorporated in the adsorber, together with suppressing cytokine production, and directly blood It is to realize a novel adsorber that can adsorb or remove the target cytokine more efficiently than the inside. It was also an object to suppress column pressure increase due to blood clogging during this operation.

In order to achieve the above object, the present invention has the following configuration.
1. Built-in water-insoluble material that adsorbs or removes leukocytes and cytokines from a blood dispersion, wherein the pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less is 30% or more and is bound with a hydrophilic amine residue. Adsorber.
2. 2. The adsorber according to 1 above, wherein the pore volume ratio is 90% or more.
3. The cytokines include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF). -Α), at least one selected from the group consisting of transforming growth factor beta (TGF-β), angiogenic growth factor (VEGF), and immunosuppressive acidic protein (IAP) Or the adsorber of 2.
4). 4. The adsorber according to any one of 1 to 3, wherein the blood cell adsorption rate from the blood satisfies the following formula when the blood of a healthy human subject is treated for 1 hour.

a / b <1
Where a: granulocyte adsorption rate (%), b: monocyte adsorption rate (%)
5. 5. The adsorber according to any one of 1 to 4, wherein the adsorption rate of granulocytes from the blood dispersion is 50% or more and the adsorption rate of monocytes is 50% or more.
6). 6. The adsorber according to any one of 1 to 5, wherein an adsorption rate of lymphocytes from the blood dispersion is 40% or less.
7). The adsorber according to any one of 1 to 6, wherein the water-insoluble material has a fibrous shape, a hollow fiber shape, or a bead shape.
8). 8. The adsorber according to 7, wherein the water-insoluble material has a fiber shape or a hollow fiber shape and has a membrane form.
9. 9. The adsorber as described in 7 or 8 above, wherein the water-insoluble material has a fiber shape or a hollow fiber shape, and the fiber diameter exceeds 3 μm.
10. 8. The adsorber according to 7 above, wherein the water-insoluble material has a bead shape and has a protruding portion having a diameter exceeding 3 μm on the surface of the material.
11. 11. The adsorber according to any one of 1 to 10, wherein an amino group in the hydrophilic amine residue is quaternized.
12 The adsorber according to any one of 1 to 11 above, which is used for a purpose of circulating blood.
13. 13. A method for obtaining a blood cell purification solution from a blood dispersion using the adsorber according to any one of 1 to 12 above.

  When the material according to the present invention is used, the leukocytes that are unnecessary and harmful to the human body, such as excessively proliferated granulocytes and monocytes, and the cytokines that transmit information to these cells are also adsorbed or removed together, so that Therefore, it is useful for blood treatment and treatment of ulcerative colitis, Crohn's disease, and autoimmune diseases known as intractable diseases. Moreover, the blood used for blood transfusion can be safely transfused by processing in advance using the adsorber of the present invention.

  Thus, according to the present invention, clogging in the blood circulation can be suppressed, and it is possible to selectively adsorb or remove white blood cells and tumor cells that are unnecessary and harmful to the human body stably in the blood for a long time. Cell removal, leukocyte removal therapy, etc. can be performed. In the present invention, blood means a blood cell component, and includes whole blood, ascites, and pleural effusion.

  Subsequently, the present invention will be described in more detail.

  The water-insoluble material incorporated in the adsorber of the present invention is an adsorbing material capable of adsorbing or removing both leukocytes and cytokines from a blood dispersion. Here, the blood dispersion liquid in the present invention refers to a liquid in which at least one of white blood cells, red blood cells, and platelets is dispersed in serum, plasma, culture medium, buffer solution or the like. In addition, the water-insoluble material in the present invention means a material that is substantially or most of insoluble in water, and a material whose weight loss when immersed in water for 60 minutes is less than 2% of the initial weight. Point to.

  Furthermore, the adsorbing material according to the present invention is characterized in that the pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less is 30% or more, and at least a hydrophilic amine residue is bonded.

  The hole in the material referred to in the present invention is a material that penetrates the material in order to form a flow path through which blood cells pass. It includes a gap between fibers and a gap between particles. When capturing leukocytes, particularly granulocytes or monocytes, which is the object of the present invention, in order to secure the blood cell flow path and prevent clogging during blood treatment, the pore diameter is constant at a ratio of 1 μm or more. It preferably occupies more than the volume, and more preferably has more than 5 μm pores.

  Further, if the pore diameter such as the above-mentioned pores is too large, it is not preferable because mechanical compressive strength and tensile strength cannot be maintained. Therefore, it is preferable that the ratio of the holes having a hole diameter of 100 μm or less occupy a certain volume or more, and it is more preferable that there are many holes of 50 μm or less in order to stabilize the shape.

  For the above reasons, it is preferable that the material incorporated in the adsorber of the present invention has a large number of pores having a pore diameter of 1 μm or more and 100 μm or less, and the proportion of such pores is occupied by all the pores of the adsorbing material in a practical state. When expressed as a ratio of the volume occupied by pores having a pore diameter of 1 μm or more and 100 μm or less to the volume (total pore volume) (referred to as pore volume ratio in the present invention), it is preferably 30% or more. When the pore volume ratio of the pores is 30% or more, it is possible to maintain the strength of the adsorbing material without clogging blood cells in the adsorbing material. Furthermore, when the pore volume ratio of such holes is 35% or more, it is possible to increase the capture rate of blood cells and the like more reliably. Furthermore, the hole volume ratio of such holes is more preferably 40% or more. In addition, especially when concentrated blood with a hematocrit value exceeding 50% is passed, the efficiency of capturing leukocytes, especially granulocytes and monocytes, is increased, and the surface area on which a large number of granulocytes and monocytes can adhere within a certain volume is increased. In order to ensure it and to prevent clogging by the captured leukocytes, it is preferable that a large number of pores having an optimum pore size exist, and it is preferable that the pores having a pore size of 1 μm or more and 100 μm or less exceed 90%. More preferably, it exceeds 92%. In terms of configuration, 100% is possible. By satisfying the above requirements, the number of pores with a small pore diameter is reduced, and as a result, an adsorber with a cell trapping ability by filtration effect lowered to the limit can be provided (selection is made when cell trapping ability by filtration effect is increased). Capturing ability decreases, that is, lymphocytes and the like may be removed excessively.) However, since there is a tendency that holes having a large hole diameter increase, in that case, it is important to provide a reinforcing structure to the adsorber. As the means, for the purpose other than the adsorption or removal of blood cells, it is conceivable to mix a mechanical strength retaining material having a diameter exceeding 10 μm, for example, to give a scrim structure. The requirement that the pore volume ratio of the pores of 100 μm or less exceeds 90% can be stably maintained. If the mechanical strength retaining material becomes thicker, the pore diameter can be kept large. However, since the cell trapping ability per unit weight of the adsorber is decreased, those having a diameter of 100 μm or less are preferably used. Furthermore, in order to set the cell capturing property per unit weight of the adsorber to a sufficient amount, the diameter is preferably 50 μm or less.

Here, in the present invention, the pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less can be obtained from a mercury intrusion curve by a mercury intrusion method. In this case, the measured value by the mercury intrusion method is a value measured in a pressure range of 1 to 2650 psia. Here, the present adsorbent material is practically used in contact with physiological saline or blood under physiological conditions, but this value needs to be measured in a deformed state as close to practical use as possible. Therefore, it is necessary to make the state of the hole as close as possible to practical use at the time of measurement.
Also, care should be taken not to change the volume of the adsorbent material due to compression when filling the measurement container.

  In addition, from the viewpoint of designing the adsorbent material and granulocytes or monocytes to make more contact during blood treatment, the fiber diameter of the fiber forming the adsorbent material (in the case of hollow fiber, hollow fiber outer diameter, the same shall apply hereinafter) .) And the diameter (size) of the protrusions on the surface of the bead particles are preferably designed from 0.5 μm to 5 μm from the viewpoint of facilitating the phagocytic ability of these blood cells. . In order to set the pore diameter of the above-mentioned material to 1 μm or more, the fiber diameter of the fiber forming the adsorbent material or the diameter (size) of the protrusion on the surface of the bead particle (hereinafter referred to as the fiber diameter) is 3 μm. Is more preferable. When the fiber diameter or the like is small, the adsorption rate or removal rate of lymphocytes may increase. In this case, the memory cell is removed. In order not to lead to a decrease in the memory cell, the lymphocyte adsorption rate is preferably 40% or less, and preferably 30% or less from the viewpoint of safety. Also from this viewpoint, it is preferable that the fiber diameter and the like exceed 3 μm because the lymphocyte capture rate can be lowered, and selectivity in the adsorption or removal of leukocytes can be imparted. In order to more efficiently adsorb or remove cells such as granulocytes and monocytes, the fiber diameter of these fibers used as a carrier made of a water-insoluble material (hereinafter, water-insoluble carrier) is 4 μm or more. Is preferred. Furthermore, from the viewpoint of more reliably suppressing the adsorption rate of lymphocytes and not leading to a decrease in the adsorption rate of granulocytes and monocytes, the fiber diameter and the like are preferably 5 μm or more. However, since the adsorption rate of granulocytes and monocytes may decrease when these diameters exceed 10 μm, it has been found that it is preferably 20 μm or less from the practical viewpoint. However, the diameter of the fiber structure mixed for the purpose other than the adsorption or removal of blood cells is not limited to this. Here, the fiber diameter in the present invention refers to the diameter of the fiber cross section. The fiber diameter or the like of the present invention or the diameter of the mechanical strength retaining material can be measured using any device such as an optical microscope, a CCD camera with a zoom, a scanning electron microscope, etc. Take 10 samples, take 1000 to 3000 times photos, measure the diameter of 100 fibers, etc., 10 places from each sample, round off the first decimal place of the average value and calculate Is possible. In particular, a scanning electron microscope (manufactured by JEOL Ltd., JSM-5400LV, Hitachi, Ltd., scanning electron microscope S-800, etc.) can be suitably used. Moreover, the diameter shown here is not applied only when the shape is a perfect circle, and is also applied when the shape is, for example, an ellipse, a rectangle, or a polygon. In these cases, the area of the figure formed by connecting the outermost layers is obtained, and the diameter of the circle corresponding to the area is obtained. However, taking a star shape with five protruding portions as an example, a figure connecting the five vertices is considered, the area is calculated, and the diameter of the corresponding circle is the diameter referred to in the present invention.

Here, the bulk density of the adsorbing material refers to the weight per fixed volume when the adsorbing material is filled in the container. When the adsorbing material is in the form of particles, the adsorbing material is placed in the container. It is desirable that the adsorber is filled with a bulk density of 0.03 g / cm ^{3 to} 0.5 g / cm ³ . Furthermore, to prevent clogging, the preferred bulk density for the flow smoothly to prevent increase in pressure loss, 0.05g / cm ³ or more 0.4 g / cm ³ or less, still more preferably a bulk density of 0.05g / Cm ³ or more and 0.3 g / cm ³ or less.

  In the present invention, the shape of the water-insoluble material that is a carrier that adsorbs leukocytes and the like is not particularly limited, but in terms of processability and pressure loss when used as a blood treatment column, the shape of fibers, hollow fibers, or beads is used. It is preferable that the fiber is a fiber that does not have a hollow fiber shape. Of course, a mixture of these may also be used. In the case of a bead shape, leukocytes and the like are captured on the particle surface by a projection having a diameter of a size described later, and the ratio of the projection portion to the entire particle surface is preferably 20% or more. .

  The form of the water-insoluble material is preferably a film form. In the present invention, the membrane form refers to a porous product having a fiber shape, a hollow fiber shape, a ribbon shape, or the like and having a large number of micropores on the surface thereof. The pore size of the micropores is preferably such that the physiologically active substances in the blood dispersion, that is, proteins, glycoproteins, polysaccharides, proteoglycans, etc., can enter the material. Includes orders.

In the adsorbing material of the present invention, the shape is particularly preferably a non-woven fabric. In that case, if the bulk density of the nonwoven fabric is too large, clogging tends to occur, and if it is too small, the shape retainability deteriorates. Therefore, the bulk density is preferably 0.02 to 0.3 g / cm ³ , more preferably 0. Those of 0.05 to 0.15 g / cm ³ are used.

  The control of the pore volume ratio is generally achieved by controlling the bulk density of the fibers in the container. When a particulate adsorbent is used, it is determined depending on its diameter. When fibers having the same fiber diameter and material are used, it is possible to control by changing the density when the fibers are assembled. In the case of using fibers, for example, the following method may be used to produce the material in the present invention, and the method is not limited to this method. That is, the diameter of the fiber to be used is 3 μm to 5 μm, an appropriate amount of fibers having a diameter of 8 μm or more are mixed, the fibers are entangled, for example, the number of punching is controlled, and the entanglement degree of the fibers is changed. Materials with a bulk density in the range can be made.

  In addition, any surface area of the adsorbing material, that is, the surface area of the capturing surface of granulocytes or monocytes can be used.

  As the basic structure of the adsorbing material used in the present invention, a material in which a functional group is immobilized on a water-insoluble carrier or a material in which a water-insoluble carrier in which a functional group is immobilized is coated on a substrate is preferable. The type of water-insoluble carrier is not particularly limited as long as it is water-insoluble and can immobilize a functional group. From the viewpoint of biocompatibility, olefin resins such as polypropylene and polyethylene are preferable, and vinyl polymers such as polyamide, polyester typified by polyethylene terephthalate, and polystyrene are preferable.

  As the adsorbing material, in order to improve the affinity with an aqueous solution or blood, the surface is preferably hydrophilic, and the material preferably has a hydrophilic coating layer. However, it is not essential to provide a hydrophilic coating layer, since it is possible to improve the affinity also by bringing it into a liquid such as ethanol in advance and then bringing it into contact with an aqueous solution. As a method for enabling the material to have a hydrophilic coating layer, the composition of the adsorbing material used is a hydrophilic water-insoluble polymer, and a hydrophilic functional group is covalently bonded to the surface of the water-insoluble polymer. Known techniques such as a method of bonding and introducing by a covalent bond (covalent bonding method) and a method of coating a hydrophilic water-insoluble polymer on a material (coating method) can be used. In the present invention, in order to maintain the function of adsorbing cytokine to the material, the covalent bonding method or the coating method is desirable among the above three methods, and the covalent bonding method is most preferable from the viewpoint of controlling the fiber diameter and the like.

  Examples of the compound used for forming a hydrophilic surface by these methods include use of a monomer having a polyalkylene glycol chain, a polymer or graft copolymer, ethylene-vinyl alcohol, and polyester. Among these, it is preferable to have a hydroxyl group in the polymer because it can prevent excessive capture of platelets.

  In the present invention, the functional group to be bound to the water-insoluble carrier is a hydrophilic amine residue (that is, a hydrophilic amine functional group), and in particular, a quaternary ammonium salt and / or a linear amino group is immobilized. Is preferred. There is no problem in immobilizing other functional groups other than the hydrophilic amine residue. In order to immobilize a quaternary ammonium salt and / or a linear amino group on a water-insoluble carrier, it is preferable to separately introduce a reactive functional group into the material. Preferred examples thereof include a halomethyl group, a haloacetyl group, and a haloacetamide. Examples include an active halogen group such as a methyl group and a halogenated alkyl group, an epoxide group, a carboxyl group, an isocyanate group, a thioisocyanate group, and an acid anhydride group. In addition to being easy to produce, the reactivity is moderately high and the quaternary ammonium salt and / or linear amino group immobilization reaction can be carried out under mild conditions, and the resulting covalent bond is chemically stable. So it is preferable.

  Here, the immobilization of the functional group means that ammonia, a primary to tertiary amino group or the like is chemically bonded to the polymer. In terms of the number of carbon atoms (hereinafter referred to as carbon number), the primary to tertiary amino groups are preferably those having 18 or less carbon atoms per nitrogen atom in order to improve the reaction rate with the reactive functional group. Further, among the primary to tertiary amino groups, a quaternary ammonium group obtained from a tertiary amino group having an alkyl group having 3 to 18 carbon atoms, particularly 4 to 14 carbon atoms, per nitrogen atom. Is excellent from the viewpoint of adsorbing cytokines. Specific examples of such tertiary amino groups include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl-hexylamine. Etc.

  The density of the immobilized quaternary ammonium salt and / or linear amino group in the material of the present invention varies depending on the chemical structure of the water-insoluble carrier or the use of the adsorber, but if it is too small, its function does not appear. On the other hand, if the amount is too large, the physical strength of the carrier after immobilization tends to be lowered and the function as an adsorbing material tends to be lowered, so that 0.01 to 2.0 moles per repeating unit of the water-insoluble carrier. More preferably, the amount is 0.1 to 1.0 mol.

  As described above, the shape of the adsorbing material is not particularly limited, but when used as a column, a fiber shape, a hollow fiber shape, or a bead shape is preferable, and a knitted fabric, a woven fabric, a nonwoven fabric, or the like using fibers. It is preferable to use a fiber structure. The material alone can be used as long as the water-insoluble carrier itself can retain its form, but if the form retainability alone is low, it can be fixed to an appropriate substrate by a method such as coating or other materials. And can be used as a single column. Such an operation such as fixing or mixing may be performed before processing into the above-described knitted fabric, fabric or the like.

  The nonwoven fabric used in the present invention may be made from a single fiber, but is particularly preferably made from a sea-island type composite fiber. That is, after making a nonwoven fabric by a known method using such a conjugate fiber, to improve the shape retention of this nonwoven fabric, and to adjust the bulk density, after needle punching, by dissolving sea components, It can be manufactured easily.

Preferred materials for the water-insoluble carrier are as described above, but are quaternary ammonium salts that can be used alone and / or polymers having a linear amino group immobilized thereon. Specific examples of polymers that can be used alone are as follows. , Poly (p-phenylene ether sulfone):-{(p-C ₆ H ₄ ) -SO _2- (p-C ₆ H ₄ ) -O-} _n -or Udel polysulfone:-{(p-C _{6 H 4) -SO 2 - (p} -C 6 H 4) -O- (p-C 6 H 4) -C (CH 3) 2 - (p-C 6 H 4) -O} n - other, _{- {(p-C 6 H} 4) -SO 2 - (p-C 6 H 4) -O- (p-C 6 H 4) -O} n -, - {(p-C 6 H 4) - _{SO 2 - (p-C 6} H 4) -S- (p-C 6 H 4) -O} n -, - {(p-C 6 H 4) -SO 2 - ( _{-C 6 H 4) -O- (p} -C 6 H 4) -C (CF 3) 2 - (p-C 6 H 4) -O} n - polysulfone type polymer represented by like, polyether Examples thereof include imide, polyimide, polyamide, polyether, polyphenylene sulfide, polystyrene, acrylic polymer, and the like, and having a reactive functional group that can fix an amino group by a covalent bond. Among these, polysulfone polymers are preferably used because of their high stability and good shape retention.

  Specific examples include chloroacetamidomethylated polystyrene, chloracetamidomethylated Udel polysulfone, chloroacetamidomethylated polyetherimide, and the like. Furthermore, those polymers that are soluble in organic solvents are particularly preferably used from the viewpoint of ease of molding.

  The adsorbing material in the present invention can be produced by a method in which a polymer having such a quaternary ammonium salt and / or a linear amino group is used as a water-insoluble carrier itself and formed into a fiber, hollow fiber or bead. In addition, fibers, hollow fibers and beads, particularly from the viewpoint of productivity, may preferably be produced by coating a substrate such as a nonwoven fabric with a polymer having a quaternary ammonium salt and / or a linear amino group. In the latter case, the polymer can be easily produced by dissolving the polymer in a solvent such as methylene chloride, tetrahydrofuran, N, N-dimethylformamide, etc., immersing the nonwoven fabric in this solution, and then evaporating the solvent.

  As a reaction solvent for immobilizing a quaternary ammonium salt and / or a linear amino group on a water-insoluble carrier, water, methanol, ethanol, isopropanol, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone and the like are preferably used.

  The adsorptive material in the present invention can enhance the selective capture of blood cells by controlling the pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less.

  When the pore volume ratio of the pores having a pore diameter of 1 μm or more and 100 μm or less is 30% or more, there is a sufficient chance of contact between the blood cells and the adsorbing material, but the supplementary action by filtration is reduced. For this reason, the foreign substance recognition power and phagocytic ability possessed by granulocytes and monocytes are significantly observed for the adsorbing material. Utilizing this property, the adsorption rate of granulocytes and monocytes can be increased compared to lymphocytes. Furthermore, by introducing a hydrophilic amine residue into the water-insoluble material, the foreign matter recognition ability of monocytes is further increased, and the monocyte adsorption rate can be increased with respect to the granulocyte adsorption rate. In contrast to granulocytes, monocytes have a higher cytokine-producing ability, and if the remaining amount in the blood is large, it is difficult to efficiently reduce the cytokine concentration in the blood. Therefore, it is desirable to use an adsorbing material having a high adsorption rate for both granulocytes and monocytes, but a / b (−) which is the ratio of the granulocyte adsorption rate a (%) to the monocyte adsorption rate b (%). ) Is preferably smaller than 1. In order to further enhance the effect, the a / b is preferably smaller than 0.9. By setting the pore volume ratio having specific pores within the above range, it is possible to effectively reduce cytokine production.

  Further, the adsorbing material in the present invention is characterized in that, by adopting the above configuration, the adsorption rate of granulocytes from the blood dispersion is 50% or more and the adsorption rate of monocytes is 50% or more. . Furthermore, it is preferable that the adsorption rate of granulocytes and monocytes is higher because the removal efficiency of blood cells can be improved.

  In the present invention, the lymphocyte adsorption rate is preferably 40% or less. Furthermore, the adsorption rate of lymphocytes is preferably 30% or less from the viewpoint of immune function.

  The evaluation of the blood cell adsorption rate in the present invention is performed using a mini-column. A cylindrical column (inner diameter 1 cm) with a packing volume of 2 ml is used to fill the adsorbing material. Heparin blood is collected (heparin concentration: 10 U / ml), and 25 ml of blood from healthy volunteers is circulated at 37 ° C. for 1 hour at a blood flow rate of 0.65 ml / min. At the start of circulation (0 minutes) and at the end of circulation (1 hour), 1 ml of blood is collected, and the number of blood cells is measured from the composition of blood cells.

  The adsorption rate in the present invention is obtained from the following equation when blood cells are taken as an example.

Adsorption rate (%) = [(blood cell amount in blood before column circulation) − (blood cell amount in blood after column circulation)] / (blood cell amount in blood before column circulation) × 100

Cytokines adsorbed by the adsorbing material in the present invention are interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10). At least one selected from the group consisting of tumor necrosis factor-α (TNF-α), transforming growth factor beta (TGF-β), angiogenic growth factor (VEGF) and immunosuppressive acidic protein (IAP) It is a cytokine. These have all been suggested to be involved in ulcerative colitis, Crohn's disease, and rheumatoid arthritis, which are pathological conditions for which leukocyte removal therapy is considered, and in recent years, their removal has shown antitumor effects. It is an expected cytokine.

  Depending on the type of cytokine to be adsorbed, the type of quaternary ammonium salt and / or linear amino group to be immobilized on the water-insoluble carrier can be appropriately selected. For example, interleukin-1 (IL-1), interleukin-6 (IL-6), transforming growth factor beta (TGF-β), angiogenic growth factor (VEGF), immunosuppressive acidic protein (IAP) ) And the like can be adsorbed by immobilizing N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine or the like on the material. In addition, interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and the like can be obtained by immobilizing tetraethylenepentamine as an amine component on a material. Adsorbability can be imparted.

  In the present invention, the evaluation of the adsorptivity or removability of these cytokines refers to those measured by enzyme immunoassay (EIA method) using natural proteins as adsorbed materials (measurement conditions at 37 ° C.). It is performed by batch adsorption for 2 hours at a temperature).

  The adsorber of the present invention can be produced by filling the column container with the adsorbing material of the present invention. As the column container, a known blood processing column container can be used. The material of the column is not particularly limited, but polypropylene, polycarbonate, nylon, polyethylene terephthalate, polyethylene, fluororesin, polyurethane, polysulfone and the like are preferably used. The column is composed of a column in which an adsorbent material is formed in a flat plate and packed with the plate, a cylindrical filter in which the adsorbent material is wound in a cylindrical shape, and a cylinder having a blood inlet and a blood outlet at both ends. A column housed in a container, a hollow cylindrical filter in which an adsorbent material is wound in a cylindrical shape, is a cylindrical container having a blood inlet and a blood outlet with both ends sealed. A column in which a blood outlet is housed in a container provided in a portion communicating with the outer peripheral portion of the hollow cylindrical filter and a blood outlet of the container in a portion communicating with the inner peripheral portion of the hollow cylindrical filter is preferable. Among them, the column using the hollow cylindrical filter allows most of the inflammatory leukocytes in the blood to be quickly adsorbed or removed by the large area nonwoven fabric on the outer periphery of the cylindrical filter, and not absorbed or removed. The remaining small amount of inflammatory leukocytes reaches the inner circumference of the cylindrical filter and is sufficiently adsorbed or removed by the nonwoven fabric of the small area, allowing efficient adsorption or removal of inflammatory leukocytes. So most preferred.

  The adsorbing material of the present invention can be used as an adsorber by filling a container having the above-described blood inlet and blood outlet. The container shape is not particularly limited as long as it has a blood inlet and a blood outlet, but to dare to give an example, a container that can be filled with a known adsorbing material in a laminated form, a cylindrical shape, a triangular prism shape, a quadrangular prism shape, Hexagonal columnar, octagonal columnar, etc. prismatic containers, containers that can be filled with a cylindrical wound of adsorbent material, or blood flows inward from the inlet arranged on the outer periphery of the cylinder and collects inside Thus, a container or the like having a shape that flows out from the central blood outlet has a good shape. There is no problem even if the container has a structure in which blood flows in the opposite direction. In addition, a container having a shape such that a cross-sectional area becomes smaller as it goes from the inlet toward the outlet can be used. If the ratio (cross-sectional area / thickness, hereinafter referred to as S / L) of the blood filtration cross-sectional area (filtration area) and blood filtration length (capture material thickness) of the adsorbent material at this time is too small, the cross-sectional area is insufficient. This is not preferable because the blood flow resistance in the adsorber may increase, and if it is too large, non-uniform flow such as a single blood flow tends to occur in the adsorber, which is caused by adhesion of granulocytes and / or monocytes. Since the capture rate is lowered, it is also not preferable. A preferable S / L range is 10 cm or more and 200 cm or less. More preferably, it is 12 cm or more and 100 cm or less.

  Here, solids larger than the pore size such as the above-mentioned fibers such as dust and blood component aggregates during washing before use enter, and aggregates of blood components aggregated in the adsorber In order to prevent the adsorber from entering the human body from the outside, a filtering material having an average pore size larger than the pore size of the adsorbing material is placed in the adsorber at a position upstream and / or downstream of the adsorbing material in the container. It is preferable to provide at the position. An average pore diameter of such a filtration material is usually 10 to 300 μm, but is not limited thereto. However, if the average pore size is less than 10 μm, clogging with blood cells on the surface of the filtering material may be caused, and pressure loss may increase, which may not be good.

  The priming amount (blood volume) of the adsorber of the present invention is not particularly limited, but is preferably as small as possible for the sake of operability and shortening the processing time. An example of a preferable priming amount is 5 to 500 ml, more preferably 10 to 200 ml, and most preferably 20 to 100 ml.

  In the present invention, the sterilization method of the adsorber may be any known method, but if an example is given, sterilization methods such as heat sterilization such as autoclave, sterilization such as γ-ray sterilization, electron beam sterilization, etc. Can be used. What is necessary is just to select suitably considering the functional group provided and the property of the water-insoluble carrier to be used.

  Specific examples of the method of using the adsorber of the present invention include equipment such as a blood bag, a blood circuit, a chamber, a clamp, a roller clamp, a drip chamber, a needle, a drip chamber with a mesh, and a blood pump tube before and after the adsorber. Any one or a plurality of these can be selected and incorporated, for example, an extracorporeal circuit or a blood transfusion circuit. The equipment incorporated in the circuit is not limited to the above. Further, a pump such as a blood pump, a liquid feed pump, or a suction pump can be incorporated in the middle of the circuit. In addition, blood can be fed by a drop due to its own weight.

Hereinafter, the present invention will be described in more detail with reference to experimental examples.
(Measurement of pore volume ratio)
Measurement was performed by a mercury intrusion method using a pore sizer 9320 manufactured by Micromeritex. The pressure range was measured as 1-2650 psia. Since it is necessary to use the value in the deformed state as close to practical as possible for the hole here, if the fiber diameter changes in saline compared to the dry state, measure the change in diameter due to swelling etc. with an optical microscope. The area was corrected by squaring the change rate, and the volume was multiplied by the cube of the change rate. Using the results obtained by measuring by this method, the pore volumes at a pressure corresponding to a pore diameter of 1 μm or more and 100 μm or less are totaled, and the ratio to the total pore volume within this pressure range, that is, the pore diameter of 1 μm or more and 100 μm or less. The pore volume ratio of the pores was determined.
(Evaluation of cytokine adsorption)
Human natural IL-1 and IL-6 were added to fetal calf serum, and these concentrations were adjusted to 500 pg / ml, respectively. An adsorbent carrier was added to this serum, and the mixture was shaken at 37 ° C. for 2 hours (usually the conditions employed in EIA measurement were used. When the total liquid volume is 1.5 ml or less, the frequency is about 10 times / second. However, if the amount of liquid is large, it is necessary to lower it). The supernatant was collected and used as a sample. The ratio of carrier: serum amount was unified as a solid-liquid ratio of 30 mg: 1 ml, and the amount of cytokine before and after shaking was determined to measure the adsorption rate. The EIA method was used for quantification, using a commercially available kit (IL-1: human IL-1β ELISA kit manufactured by R & D System, IL-6: manufactured by Kamakura Technoscience), and the adsorption rate was calculated by the following formula. .

Adsorption rate (%) = [(amount of cytokine in blood before column circulation) − (amount of cytokine in blood after column circulation)] / (amount of cytokine in blood before column circulation) × 100
(Measurement of blood cell count)
The quantification of the number of blood cells in the body fluid and the measurement of the hematocrit value were performed using Sysmex XT-1800Vi. The adsorption rate of granulocytes and monocytes was calculated by the following formula.

Adsorption rate (%) = [(blood cell amount in blood before column circulation) − (blood cell amount in blood after column circulation)] / (blood cell amount in blood before column circulation) × 100
(Measurement of fiber diameter, etc.)
Ten small sample samples are collected at random from the adsorbent material, and 1000 to 3000 times photographs are taken using a scanning electron microscope S-800 manufactured by Hitachi, Ltd. 10 pieces from each sample, including 100 fibers in total. Diameters were measured and calculated by rounding off the first decimal place of the average value.
[Production example]
(Nonwoven fabric)
A sea island composite fiber of 36 islands, in which the island is further composed 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 90wt%, Polypropylene 10wt%
Sea component: ethylene terephthalate unit as the main repeating unit, copolymerized component containing 3% by weight of 5-sodiumsulfoisophthalic acid as a copolymer component (weight ratio); core: sheath: sea = 40: 40: 20
A sheet-like material composed of 70% by weight of this fiber and 30% by weight of polypropylene fiber having a diameter of 25 μm was produced, and then a nonwoven fabric was obtained by needle punching. Next, this non-woven fabric was treated with an aqueous solution of sodium hydroxide (3 wt%) at 90 ° C. to form a sea component “ethylene terephthalate unit as a main repeating unit, and a copolymer containing 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component. By dissolving “polyester”, a nonwoven fabric having a core-sheath fiber diameter of 4 μm and a bulk density of 0.05 g / cm ³ (total basis weight 200 g / m ² ) was produced (nonwoven fabric).
(Intermediate)
Next, 3 g of paraformaldehyde was dissolved at 20 ° C. in a mixed solution of 600 mL of nitrobenzene and 390 mL of sulfuric acid, then cooled to 0 ° C., and 75.9 g of N-methylol-α-chloroacetamide was added and dissolved at 5 ° C. or lower. did. 5 g of the non-woven fabric was immersed in this and allowed to stand at room temperature for 2 hours. Thereafter, the fiber was taken out, placed in a large excess of cold methanol and washed. The fiber was thoroughly washed with methanol, then washed with water, and dried to obtain 6.2 g of α-chloroacetamidomethylated polystyrene fiber (intermediate).
(Adsorption material)
5 g of the intermediate was immersed in a solution of 50 g of N, N-dimethylhexylamine and 8 g of potassium iodide in 360 mL of dimethylformamide (DMF) and heated in a 85 ° C. bath for 3 hours. The fiber was washed with isopropanol, immersed in 1000 ml of 1 mol / L saline solution for 3 hours, washed with water, and dried under vacuum to obtain 8.1 g of dimethylhexylammoniumated fiber (adsorbing material 1).
[Example 1]
Heparin blood was collected (10 U / ml) from 50 ml of healthy volunteer blood, and human natural IL-1 and IL-6 were dissolved therein so that the concentration was 500 pg / ml. The hematocrit value was 45%.

  150 mg of adsorbent material 1 is taken and packed into a cylindrical column with a packing volume of 2 ml (inner diameter 1 cm), and after circulating 25 ml of the blood at 37 ° C. for 1 hour at a blood flow rate of 0.65 ml / min, the composition of the blood cells is automatically A blood analyzer was used and the amounts of IL-1 and IL-6 were quantified by the EIA method. By column circulation, the number of lymphocytes in the blood decreased by 12.5%, granulocytes decreased by 76%, monocytes decreased by 81%, and IL-1 and IL-6 decreased by 21% and 28%, respectively. At this time, the value of granulocyte removal rate / monocyte removal rate was 0.94. Separately, as a result of cytokine adsorption evaluation, the adsorption rates of IL-1 and IL-6 were 51% and 78%, respectively.

A column with 150 mg of the adsorbing material 1 and an internal volume of 2 ml was packed, and the pore volume ratio was measured with the solution channel opened. The pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 35%.
[Example 2]
Heparin blood was collected (10 U / ml) from 50 ml of healthy volunteer blood, and human natural IL-1 and IL-6 were dissolved therein so that the concentration was 500 pg / ml. The hematocrit value was 47%.

  250 mg of adsorbent material 1 was taken and packed into a cylindrical column with a packing volume of 2 ml (inner diameter 1 cm), and after circulating 25 ml of the blood at 37 ° C. for 1 hour at a blood flow rate of 0.65 ml / min, the composition of the blood cells was automatically A blood analyzer was used and the amounts of IL-1 and IL-6 were quantified by the EIA method. As a result of column circulation, the number of lymphocytes in the blood decreased by 32.8%, granulocytes decreased by 88%, monocytes decreased by 92%, and IL-1 and IL-6 decreased by 30% and 38%, respectively. At this time, the value of granulocyte removal rate / monocyte removal rate was 0.96. Separately, as a result of cytokine adsorption evaluation, the adsorption rates of IL-1 and IL-6 were 53% and 88%, respectively.

The column of the adsorbing material 1 was packed in 250 mg of an internal volume of 2 ml, and the pore volume ratio was measured with the solution flow path opened. The pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 77%.
[Example 3]
Heparin blood was collected (10 U / ml) from 100 ml of healthy volunteer blood. Human natural IL-1 and IL-6 were dissolved therein so as to be 500 pg / ml, and the following examination was performed. The hematocrit value was 48%.

  150 mg of the adsorbent material 1 was sampled and packed into a cylindrical column with a packing volume of 2 ml (inner diameter 1 cm), and after circulating 25 ml of the blood at 37 ° C. for 1 hour at a blood flow rate of 0.65 ml / min, the composition of the blood cells was automatically A blood analyzer was used and the amounts of IL-1 and IL-6 were quantified by the EIA method. By column circulation, the number of lymphocytes in the blood decreased by 9.8%, granulocytes decreased by 72%, monocytes decreased by 76%, and IL-1 and IL-6 decreased by 19% and 22%, respectively. At this time, the value of granulocyte removal rate / monocyte removal rate was 0.95. Separately, as a result of cytokine adsorption evaluation, the adsorption rates of IL-1 and IL-6 were 46% and 71%, respectively.

150 mg of the adsorbent material 1 was sampled and packed in a column with an internal volume of 2 ml, and the pore volume ratio was measured with the solution flow path opened. The pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 92%.
[Example 4]
Heparin blood was collected (10 U / ml) from 100 ml of healthy volunteer blood. Human natural IL-1 and IL-6 were dissolved therein so as to be 500 pg / ml, and the following examination was performed. The hematocrit value was 43%.

  200 mg of the adsorbent material 1 was sampled and packed into a cylindrical column with a packing volume of 2 ml (inner diameter 1 cm), and after circulating 25 ml of the blood at 37 ° C. for 1 hour at a blood flow rate of 0.65 ml / min, the blood cell composition was automatically A blood analyzer was used and the amounts of IL-1 and IL-6 were quantified by the EIA method. Column circulation reduced the number of lymphocytes in the blood by 10.8%, granulocytes by 75%, monocytes by 82%, and IL-1 and IL-6 by 19% and 25%, respectively. It was. At this time, the value of granulocyte removal rate / monocyte removal rate was 0.91. Separately, as a result of cytokine adsorption evaluation, the adsorption rates of IL-1 and IL-6 were 45% and 73%, respectively.

250 mg of the adsorbing material 1 was taken and packed in a column with an internal volume of 2 ml, and the pore volume ratio was measured with the solution flow path opened. The pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 97%.
[Comparative Example 1]
100 mg of the adsorbent material 1 was taken and packed in a cylindrical column with a packing volume of 2 ml (inner diameter 1 cm), and the test was performed under the same conditions as in Example 1 (blood volume was 25 ml). The column decreased the number of lymphocytes in the blood by 8.4%, granulocytes by 43%, monocytes by 48%, and IL-1 and IL-6 by 13% and 18%, respectively. Separately, as a result of cytokine adsorption evaluation, the adsorption rates of IL-1 and IL-6 were 49% and 78%, respectively. At this time, the value of granulocyte removal rate / monocyte removal rate was 0.89.

  100 mg of the adsorbent material 1 was sampled and packed in a column with an internal volume of 2 ml, and the pore volume ratio was measured with the solution channel opened. The pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less was 29%. The adsorption rate of granulocytes and monocytes was less than 50%, and the adsorption efficiency was poor.

  From the above results, the adsorber according to the present invention having a pore volume ratio of 30% or more with pore diameters of 1 μm or more and 100 μm or less can adsorb granulocytes and monocytes in blood with high efficiency, and also includes cytokines. It was found that it can adsorb with high efficiency.

  The adsorber according to the present invention can efficiently adsorb and / or remove leukocytes, inflammatory and immunosuppressive cytokines in blood, and reduce the adsorption rate and / or removal rate of useful components in blood. It can be provided for leukocyte removal therapy.

  The material in the present invention is in the form of a petri dish, a bottle, a membrane, a fiber, a hollow fiber, a granular material or a molded product such as an assembly using these, and is used for an affinity chromatography column, a therapeutic blood column, particularly an extracorporeal circulation. It can be suitably used as a column.

Claims (11)

Built-in water-insoluble material that adsorbs or removes leukocytes and cytokines from a blood dispersion, wherein the pore volume ratio of pores having a pore diameter of 1 μm or more and 100 μm or less is 30% or more, and is bound with a hydrophilic amine residue,
An adsorber characterized in that when blood from healthy human subjects is treated for 1 hour, the blood cell adsorption rate from the blood satisfies the following formula.
a / b <1
Here, a: granulocyte adsorption rate (%), b: monocyte adsorption rate (%).   The adsorber according to claim 1, wherein the pore volume ratio is 90% or more.   The cytokines include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF). -Α), transforming growth factor beta (TGF-β), angiogenic growth factor (VEGF), and immunosuppressive acidic protein (IAP). The adsorber according to 1 or 2.   The adsorber according to any one of claims 1 to 3, wherein an adsorption rate of granulocytes from the blood dispersion liquid is 50% or more and an adsorption rate of monocytes is 50% or more.   The adsorber according to any one of claims 1 to 4, wherein an adsorption rate of lymphocytes from the blood dispersion is 40% or less.   The adsorber according to any one of claims 1 to 5, wherein the water-insoluble material has a fibrous shape, a hollow fiber shape, or a bead shape.   The adsorber according to claim 6, wherein the water-insoluble material has a fiber shape or a hollow fiber shape and has a membrane form.   The adsorber according to claim 6 or 7, wherein the water-insoluble material has a fiber shape or a hollow fiber shape, and the fiber diameter exceeds 3 µm.   The adsorber according to claim 6, wherein the water-insoluble material has a bead shape and has a protruding portion having a diameter exceeding 3 μm on the surface of the material.   The adsorber according to any one of claims 1 to 9, wherein an amino group in the hydrophilic amine residue is quaternized.   The adsorber according to any one of claims 1 to 10, wherein the adsorber is used for a purpose of circulating blood.