JP4983070B2 - Adsorbent and extracorporeal circulation column - Google Patents

Description

    The present invention relates to an adsorbent and a blood treatment column suitably used for so-called leukocyte removal therapy, which removes leukocytes from blood.

  The blood contains various components such as blood cells, cytokines and other humoral components, and these blood components play an important role in regulating the balance of immunity in the body.

  Endotoxins represented by lipopolysaccharide exhibit various biological activities such as fever, blood pressure reduction, intravascular coagulation, and Hageman factor activation in the blood. Particularly in the clinical setting, for example, severe sepsis caused by endotoxin mixed in patient blood after surgery, or cancer necrosis factor, interleukin 1, interleucine 6, interferon, etc. from leukocytes stimulated by endotoxin, etc. It is known that these excess cytokines have an adverse physiological effect due to the release of various cytokines and acid peroxides. Various cytokines such as cancer necrosis factor, interleukin 1, interleucine 6, and interferon from leukocytes, and acid peroxide due to endotoxins mixed in the blood of patients, particularly endotoxins mixed in the blood of serious patients Are released, and these excess cytokines are known to have adverse physiological effects.

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

    In these column carriers, removal of leukocytes themselves, removal of granulocytes themselves, and removal of cytokines themselves have been made efficient, but humoral factors to which blood cells respond to information cannot be removed. Normalization of the remaining cells becomes insufficient.

    Columns designed to adsorb leukocytes and toxins at the same time (Patent Documents 5 and 6) have also been developed, but the remaining bioactive substance adsorbed by cells such as cytokines cannot be removed. Cell normalization is insufficient.

Although there is an invention of a leukocyte-removing carrier that specifies that the zeta potential is 0 mV or more (Patent Document 7), the disclosure of blood cell removal is limited and normalization of the remaining cells becomes insufficient.
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 JP-A-6-142196

An object of the present invention is to remove cells such as granulocytes and monocytes, and to prevent cytokines that promote activation of the remaining cells from remaining in the remaining liquid component. For this purpose, it was considered that these problems can be solved by giving the adsorption carrier a function capable of removing cytokines that increase abnormally simultaneously with cell removal.

  The present inventors adsorb and remove endotoxin in the body fluid that is present in the body fluid and cause cytokine release, endotoxin adhering to the surface of granulocytes and monocytes, or production of excess cytokine by endotoxin. It has been found that improving the state of endotoxin-containing blood by prevention is an important issue. In the present invention, endotoxin in a body fluid is directly adsorbed by an adsorbent, and endotoxin adhering to leukocyte components such as granulocytes or monocytes is adhered to the capturing material. It is intended to be removed from the blood indirectly. Furthermore, the cytokine caused by the endotoxin is also removed to prevent an increase in cytokine concentration in the blood.

  That is, the present invention eliminates endotoxin, cells such as granulocytes and monocytes, and prevents cytokines that promote activation of the remaining cells from remaining in the remaining liquid components. In order to solve this problem, the present invention provides a highly functional material that can be suitably used for adsorption of both cells and endotoxins and cytokines, and a highly functional blood processing column using the same. It is intended to provide.

    In order to achieve the above object, the present invention has the following configuration.

    1. An adsorbent having a zeta potential of −20 mV or more and adsorbing granulocytes, monocytes and cytokines in blood.

    2. 2. The adsorber according to 1 above, wherein the adsorption rate of granulocytes is 50% or more and the adsorption rate of monocytes is 50% or more.

    3. 2. The adsorbent according to 1 or 2 above, wherein the adsorption rate of lymphocytes is 40% or less.

    4). The cytokine is interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), TNF-α, Any one of the above 1 to 3, which is at least one selected from the group consisting of Gross Factor Beta (TGF-β), Angiogenic Growth Factor (VEGF) and Immunosuppressive Acidic Protein (IAP) The adsorbent described.

    5). 5. The adsorbent according to any one of 1 to 4 above, wherein a quaternary ammonium salt and / or a linear amino group is bonded to a water-insoluble carrier.

    6). 2. The adsorbent according to 1 above, wherein the adsorbent has a zeta potential of −15 mV or more and has a lipopolysaccharide (LPS) adsorption capacity of 90% or more in 1 vol% fetal calf serum (FCS) -dissolved physiological saline.

    7). The adsorbent according to any one of 1 to 6, wherein the adsorbent has a shape selected from fibers, membranes, hollow fibers, and beads.

    8). 8. The adsorbent according to any one of 1 to 7 above, comprising a water-insoluble carrier formed by bonding a functional group.

    9. 9. The adsorbent according to 8 above, wherein the shape of the water-insoluble carrier is a shape selected from fibers or hollow fibers having a fiber diameter exceeding 3 μm, and beads having a surface protrusion having a diameter exceeding 3 μm.

    10. 9. The shape of the water-insoluble carrier is a shape selected from fibers or hollow fibers having a fiber diameter of 4 to 8 μm, and beads having a diameter of protrusions on the surface of the beads of 4 to 8 μm. Adsorbent.

    11. The shape of the water-insoluble carrier is a shape selected from fibers or hollow fibers having a fiber diameter of 4.5 to 8 μm, and beads having a diameter of protrusions on the surface of the bead particles of 4.5 to 8 μm. 9. The adsorbent as described in 8 above.

    12 The adsorbent according to any one of 9 to 11, wherein the shape of the water-insoluble carrier further includes fibers or hollow fibers having a fiber diameter of 10 to 50 µm.

    13. 13. The adsorbent according to any one of 8 to 12 above, wherein a quaternary ammonium salt and / or a linear amino group is bonded to a water-insoluble carrier.

    14. The quaternary ammonium salt is at least one selected from N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine and tetraethylenepentamine, Adsorbent as described in 4.

    15. 15. The adsorbent according to any one of 1 to 14 above, which is used for leukocyte removal therapy.

    16. A blood processing column comprising a container filled with the adsorbent according to any one of 1 to 15 above.

    17. 17. The blood processing column according to 16, wherein blood is circulated.

    18. 18. The blood processing column as described in 16 or 17 above, which is used for leukocyte removal therapy.

    19. The blood processing column according to any one of 16 to 18, which shows an increase in the number of lymphocytes and a decrease in the number of granulocytes after 150 to 180 hours from the extracorporeal circulation with a living organism, compared to before the extracorporeal circulation.

  According to the present invention, leukocytes unnecessary for the human body such as excessively proliferated granulocytes and monocytes and cytokines that transmit information to these cells are simultaneously removed, and human bodies such as excessively proliferated granulocytes and monocytes It is useful for blood treatment and treatment in ulcerative colitis, Crohn's disease, autoimmune disease, etc. by simultaneously removing unnecessary white blood cells, cytokines that transmit information to these cells, and LPS that activates white blood cells Adsorbents and blood processing columns are provided.

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

  A preferred embodiment as the adsorbent of the present invention is an adsorbent having a zeta potential of −15 mV or more.

  As a basic configuration of the adsorbent, 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 water-insoluble carrier used in the present invention is not particularly limited as long as it is insoluble in water and can immobilize a functional group. From the viewpoint of biocompatibility, olefin-based resins such as polypropylene and polyethylene are preferred, and polyamides and polyesters typified by polyethylene terephthalate are preferred, but caution is required for hydrolysis.

In the adsorbent of the present invention, the zeta potential is used for recognizing sialic acid and phosphoric acid on glycoproteins on the cell surface, and for recognizing sialic acid and phosphoric acid on sugar chains bound to cytokines and the like. Higher is preferable. Conventional polymer materials such as polypropylene, polyethylene, and polyethylene terephthalate have a negative zeta potential and are approximately -30 mV. Therefore, the present inventors fixed a specific functional group such as a quaternary ammonium salt and / or a linear amino group on a water-insoluble carrier, and set the zeta potential to -20 mV or higher. The inventors have found that the characteristics are good and have reached the present invention.
Furthermore, if the zeta potential is −15 mV or more, the adsorption property for lipopolysaccharide (LPS) is further improved, which is preferable, and if it is −10 mV or more, the effect is higher, and −2 mV or more is more preferable.

  In the present invention, it is preferable that the adsorbent has the ability to adsorb LPS as well as the ability to adsorb granulocytes, monocytes and cytokines. LPS is a toxin generally possessed by gram-negative bacteria as described later. LPS has been found to bind to and activate receptor proteins such as TLR-4 on leukocytes. By removing granulocytes and / or monocytes and cytokines, abnormal conditions caused by these can be reduced. However, if LPS generated from bacteria that have invaded from inflammatory sites or ulcer sites remains, the patient's condition In spite of having shifted to a state close to a normal state, it may fall into an abnormal state again. By providing the adsorbent of the present invention with a preferable mechanism for removing LPS at the same time as the ability to adsorb leukocytes and cytokines, it can be efficiently used for blood treatment and treatment of ulcerative colitis, Crohn's disease, autoimmune disease and the like. It can be usefully used. Further, by using such a multifunctional adsorbent, it is possible to make the therapeutic column compact.

  In the present invention, the zeta potential refers to the zeta potential on the surface of the adsorbent. The surface zeta potential can be obtained by calculation by measuring the streaming potential and the pressure applied to flow the liquid and the specific conductivity of the liquid with a streaming potential measuring device. The adsorbent of the present invention preferably has a zeta potential of −20 mV or more, more preferably −15 mV or more, and particularly preferably −2 mV or more. The upper limit is preferably 10 mV or less from the viewpoint of preventing red blood cell hemolysis and the like.

    In the present invention, the shape of the water-insoluble carrier is not particularly limited, but is preferably a fiber, membrane, hollow fiber, or bead shape from the viewpoints of processability and pressure loss when used as a blood treatment column. Of course, these combinations may be used.

  Further, the adsorbent in the present invention has the ability to adsorb granulocytes, monocytes and cytokines in blood, in particular, the adsorption rate of granulocytes from blood is 50% or more, and the adsorption rate of monocytes Is preferably 50% or more. Here, the adsorption rate in the present invention, when taking blood cells as an example, measures the amount of blood cells before and after passing through a column packed with an adsorbent once with a blood cell analyzer, It is obtained from the formula. Conditions such as the column size, shape, blood passage speed, and the like can be determined by the method exemplified in the examples, and there is no problem as long as the conditions have results equivalent to this.

Adsorption rate (%) = [(blood cell volume in the blood before passing through the column) − (blood cell volume in the blood after passing through the column)] / (blood cell volume in the blood before passing through the column) × 100
The adsorbent of the present invention has an action of adsorbing blood cells, but may further have a filtering action. In this case, the calculation target of the adsorption rate includes not only blood cells removed from blood by adsorption but also blood cells removed by filtration.

  In order to adsorb and remove cells such as granulocytes and monocytes, the adsorbent of the present invention is preferably used as a water-insoluble carrier. Among them, the fiber diameter of fibers, hollow fibers, or the diameter of protrusions on the surface of bead particles It is preferable that (size) or the like (hereinafter referred to as fiber diameter or the like) exceeds 3 μm. In particular, if the diameter is smaller than this, adsorption / removal of lymphocytes increases, which leads to removal of memory cells, which is not preferable. However, with respect to the fiber diameter, in order to suppress the adsorption removal rate of lymphocytes, a more preferable range is 4 μm or more, and a further preferable range is 4.5 μm or more. Furthermore, from the viewpoint of further suppressing the removal rate of lymphocytes and not leading to a decrease in the removal rate of granulocytes and monocytes, it may be more preferable to set the fiber diameter to 5 μm or more. However, when the fiber diameter exceeds 8 μm, the removal rate of granulocytes and monocytes tends to decrease, and when the fiber diameter is 10 μm or more, the removal rate of granulocytes and monocytes decreases. Absent. From the practical viewpoint, the fiber diameter is preferably 20 μm or less.

  On the other hand, for the purpose other than the removal of blood cells, that is, the purpose of maintaining the strength of the adsorbent at a certain level or more, the fibers (referred to as fiber A) are mixed with a fiber having a larger diameter as a fiber structure. (Referred to as fiber B). The diameter of the fiber B is not limited to this, and the diameter of the fiber B is preferably 10 μm to 50 μm. When the thickness is less than 10 μm, the effect of maintaining the strength, which is the main purpose of mixing the fibers B, may not be expected. When it exceeds 50 micrometers, mixing with the fiber A may become difficult.

  The removal of lymphocytes is preferably 40% or less because it is difficult to reduce the memory cell, and more preferably 30% or less from the viewpoint of safety.

  The adsorbent of the present invention is preferably one in which a quaternary ammonium salt and / or a linear amino group is immobilized as a functional group on the water-insoluble carrier. Examples of reactive functional groups for immobilizing a quaternary ammonium salt and / or a linear amino group on a water-insoluble carrier include active halogen groups such as a halomethyl group, a haloacetyl group, a haloacetamidomethyl group, and a halogenated alkyl group, Epoxide groups, carboxyl groups, isocyanic acid groups, thioisocyanic acid groups, acid anhydride groups, and the like can be mentioned. In particular, active halogen groups, especially haloacetyl groups, are easy to produce and have moderate reactivity. In addition, the quaternary ammonium salt and / or the linear amino group can be immobilized under mild conditions, and the resulting covalent bond is chemically stable.

  The functional group to be immobilized is preferably a quaternary ammonium salt and / or a linear amino group, which means ammonia and those having a primary to tertiary amino group chemically bonded to the polymer. . Furthermore, as the primary to tertiary amino groups, those having 18 or less carbon atoms per nitrogen atom are preferable for improving the reaction rate. Further, among the primary to tertiary amino groups, a quaternary ammonium group formed by bonding a tertiary amino group having an alkyl group having 3 to 18 carbon atoms, particularly 4 to 14 carbon atoms per nitrogen atom. Is immobilized from the viewpoint of cytokine adsorption. Specific examples of such tertiary amino groups include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl- And hexylamine. Examples of the compound having a linear amino group include tetraethylenepentamine.

  The density of the quaternary ammonium salt and / or linear amino group bond in the present invention varies depending on the chemical structure and use of the water-insoluble carrier, but if it is too small, its function tends not to be expressed, while if too large, Further, since the physical strength of the carrier after immobilization is deteriorated and the function as an adsorbent tends to be lowered, the density is 0.01 to 2.0 mol, more preferably 0.8 mol per repeating unit of the water-insoluble carrier. 1-1.0 mol is good. In addition, as a method for immobilizing a quaternary ammonium salt and / or a linear amino group (quaternization), a reaction using potassium iodide as a catalyst is often used. It is possible.

  On the other hand, the adsorbent of the present invention may be one in which a functional group is immobilized on a water-insoluble carrier as described above, or in place of this, a hydrophobic site is added.

Cytokines adsorbed by the adsorbent of 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. They are,
These include cytokines and the like that are alleged to be involved in the pathology of ulcerative colitis, Crohn's disease, rheumatoid arthritis, etc. for which leukocyte removal therapy is considered.

  A quaternary ammonium salt and / or a linear amino group to be immobilized may be appropriately selected depending on the kind of cytokine to be adsorbed. 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 and the like. Also, interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), etc. are adsorbed by immobilizing tetraethylenepentamine as an amine component. Can be granted. In addition, a plurality of types of functional groups can be combined and immobilized. For example, both a quaternary ammonium salt and a linear amino group can be used. Use of a plurality of types of functional groups in combination is preferable because the types of cytokines to be adsorbed can be widened, and there are advantages such as enhancing desired cytokine adsorption characteristics.

  In the present invention, the evaluation of the adsorptive and desorbing properties of these cytokines refers to those measured by EIA method (enzyme immunoassay) using natural proteins (conditions are shaken at 37 ° C. for 2 hours, Batch adsorption.) For example, it is preferable that IL-1 and IL-6 have an adsorption rate of 40% or more and 50% or more, respectively, in the batch adsorption method described in the examples. Furthermore, in order to reduce the influence on the remaining cells, it is preferable that the adsorption rate is 50% or more and 60% or more.

  As described above, the shape of the adsorbent is not particularly limited. However, when used as a column, beads, fibers, membranes, hollow fibers, knitted fabrics made of fibers, fiber structures such as woven fabrics and nonwoven fabrics are used. preferable. If the water-insoluble carrier can retain its form by itself, it can be used alone. If the form-retaining property is low, it can be fixed to an appropriate substrate by a method such as coating, or mixed with other adsorbents to form one It can also be used as a column. Operation such as immobilization or mixing may be performed before processing into the above shape.

In the adsorbent 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, it tends to be clogged, and conversely if it is too small, the shape retainability deteriorates, so 0.02 g / cm ³ or more is preferable, and 0.05 or more is more preferable. The upper limit is preferably at 0.15 g / cm ³ or less, and more preferably 0.15 g / cm ³ or less is used.

  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, this nonwoven fabric is subjected to a needle punch to improve shape retention and to adjust the bulk density, and then by dissolving sea components, It can be manufactured easily.

Preferred materials for the water-insoluble carrier are hydrophobic fibers, that is, polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate and polybutylene terephthalate, and fluorinated polymers such as Teflon. It can also be achieved by adding various alkyl groups by modification. Specific examples of a quaternary ammonium salt that can be used alone and / or a polymer suitable for immobilizing a linear amino group include poly (p-phenylene ether sulfone):-{(p-C ₆ H ₄ ). _{-SO 2 - (p-C 6} H 4) -O-} n- and _{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 - (p-C 6 H 4) -O- (p-C 6 H 4) -C (CF 3) 2 - ( p-C ₆ H ₄ ) —O} _n — and other polysulfone polymers, polyetherimide, polyimide, polyamide, polyether, polyphenylene sulfide, polystyrene, acrylic polymer, and the like, and the amino group is immobilized by a covalent bond Those having reactive functional groups that can be used are used. Of these, polysulfone polymers are preferably used because of their high stability and good shape retention.
Specific examples include chloracetamidomethylated polystyrene to which a reactive functional group is bonded, chloracetamidomethylated Udel polysulfone, chloracetamidomethylated 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 adsorbent of the present invention is produced by forming a polymer having such a quaternary ammonium salt and / or a linear amino group by forming a water-insoluble carrier itself into fibers, hollow fibers and beads, fibers, hollow fibers and beads, In particular, from the viewpoint of productivity, a substrate such as a non-woven fabric is preferably coated with a polymer having a quaternary ammonium salt and / or a linear amino group. At that time, the polymer is 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.

  In addition, the adsorbent and the blood treatment column of the present invention preferably have the activity of blood coagulation factor XIII (coagulation factor XIII) not reduced when blood treatment is performed in consideration of safety. Particularly in patients with ulcerative colitis and Crohn's disease, blood coagulation factor XIII tends to decrease, and there is a risk of falling into a bleeding tendency due to lack of this factor. If the adsorption rate of blood coagulation factor XIII is 30% or less, it can be used safely, but it is more preferably 20% or less. The adsorption rate is calculated by calculating the ratio of the amount of carrier used to the amount of blood actually processed (in the present invention, blood means whole blood, plasma, serum, ascites, pleural effusion) It can be determined from blood coagulation factor XIII activity before and after shaking at 37 ° C. for 1 hour using plasma prepared from blood collected from healthy volunteer blood. The blood coagulation factor XIII activity can be measured by a synthetic substrate method outsourced to SRL.

  The adsorbent and blood treatment column of the present invention may have a lipopolysaccharide (LPS) adsorption capacity of 90% or more in 1 vol% fetal calf serum (FCS) -dissolved physiological saline.

  By having lipopolysaccharide adsorbing ability in this way, it is possible to prevent excessive cytokine production by lipopolysaccharide such as endotoxin that is mixed into the patient's blood after surgery or from inflammatory or ulcerous sites. Adsorbents that can effectively adsorb endotoxin itself, and can provide preventive and therapeutic effects on fever, shock, intravascular coagulation, lymphocyte activation, etc. derived from the biological activity of endotoxin, and are particularly useful in immunostimulation therapy It becomes. Furthermore, it is preferable in that not only the ability to adsorb granulocytes, monocytes and cytokines but also the ability to adsorb lipopolysaccharide allows simultaneous treatment with one adsorbent.

  Lipopolysaccharide means a molecule containing the structure of lipid A, and typical LPS that can be present in blood includes toxins possessed by gram-negative bacteria.

  Endotoxin is known to cause excessive release of various cytokines such as cancer necrosis factor, interleukin 1, interleukin 6, and interferon, and acid peroxide from leukocytes when present in excess in blood. The adsorbent and blood treatment column of the present invention are further presumed to be caused by activation of lymphocytes, particularly Th1 type lymphocytes, by imparting LPS adsorption capacity to the adsorbent of the present invention. Interferon gamma production can be improved and is useful in immunostimulation therapy. Although the detailed mechanism of the activation of such lymphocytes is unknown, it is presumed to be caused by the contact of blood cells in the treated blood with the adsorbed LPS.

  The amount of LPS adsorbed can be measured with a Toxinometer manufactured by Wako Pure Chemical Industries. The LPS remaining in the supernatant obtained by mixing a specified amount of LPS in 1% FCS-added physiological saline and incubating in a 37 ° C. water bath for 4 hours, was similarly measured with a toxinometer. The removal amount and removal rate can be determined from the difference and ratio with the LPS in the medium. The removal amount is desirably 100 pg / mg or more, more preferably 200 pg / mg or more. The ability of adsorbing granulocytes and / or monocytes in the body fluid of the present invention and cytokines simultaneously and having LPS adsorption ability in 1% FCS-dissolved physiological saline can be achieved by incorporating different adsorbents into one column. This can be realized by combining separate adsorbents of cassette type, but in the latter case, compactness is particularly easy to sacrifice, and it is more convenient to use a single adsorbent. In addition, there is no need to think in a limited manner, and it is possible to appropriately select a form according to necessity.

The blood processing column of the present invention can be produced by filling the column container with the adsorbent of the present invention. As the column container, a known blood processing column container can be used. The column structure includes a column in which an adsorbent is formed in a flat plate shape and is packed in a stacked manner, a cylindrical filter in which the adsorbent is wound in a cylindrical shape, and a cylinder having a blood inlet and a blood outlet at both ends. A column and a hollow cylindrical filter made by adsorbing material in a cylindrical shape are housed in a cylindrical container having a blood inlet and a blood outlet with both ends sealed. Preferably, the column is provided in a portion where the blood outlet of the container communicates with the outer peripheral portion of the hollow cylindrical filter, and the blood outlet of the container is provided in a portion which communicates with the inner peripheral portion of the hollow cylindrical filter. Among them, the column using a cylindrical hollow filter is
Most of the inflammatory leukocytes in the blood are quickly removed by the large area nonwoven fabric on the outer periphery of the cylindrical filter, and the small amount of inflammatory leukocytes remaining without being removed also reaches the inner periphery of the cylindrical filter. Thus, even a non-woven fabric having a small area is sufficiently removed, and efficient inflammatory leukocyte removal is possible. In particular, when the adsorbent of the present invention is made of a non-woven fabric, in preparing an extracorporeal circulation column for cancer treatment, it has a two-layer structure with a net as described later, preferably a structure in which the net is wrapped with a non-woven fabric. The form retainability can be enhanced by, for example, winding in a cylindrical shape.

  The blood processing column of the present invention can be used for leukocyte removal therapy and immunostimulation therapy. In addition, as described later, when 150 to 180 hours have passed after completion of extracorporeal circulation with a living body using the blood processing column of the present invention, the number of leukocytes in blood, particularly granulocytes, is decreased compared with that before extracorporeal circulation. The number of balls increases. This suggests that the blood processing column of the present invention has a function of correcting the immune state. In particular, it is known that patients with advanced cancer have increased granulocytes and decreased lymphocytes, and their immune status is suppressed. It will be corrected. This effect does not last for a long time after extracorporeal circulation, and generally after about one week, granulocyte increase and lymphocyte decrease tend to appear again. Therefore, treatment once a week is desired.

  The various adsorbents of the present invention described above can be used as blood treatment columns such as extracorporeal circulation columns. When used as an extracorporeal circulation column, although it depends on the amount of adsorbent to be filled in the column container and the blood circulation rate, it is usually 150 hours from the end time after the extracorporeal circulation with the living body for one to two hours. After ˜180 hours, an increase in the number of lymphocytes and a decrease in the number of granulocytes can be realized as compared with before the extracorporeal circulation. Therefore, the adsorbent and blood processing column of the present invention are useful in leukocyte removal therapy and immunostimulation therapy.

    Hereinafter, the present invention will be described in more detail with reference to experimental examples.

[Example 1 and Comparative Example 1]
(Measurement of zeta potential)
The surface zeta potential was obtained by calculation by measuring the streaming potential, the pressure applied to flow the liquid, and the kinetic dielectric constant of the liquid with a streaming potential measuring device (ZP-10B (manufactured by Shimadzu Corporation)). . The fluid was measured using 1 mM KCl aqueous solution at pH 6 ± 1 and temperature 20 ± 5 ° C.

(Evaluation of cytokine adsorption)
Human natural IL-1 and IL-6 were added to fetal calf serum and adjusted to 500 pg / ml. An adsorbent carrier was added to the serum, shaken at 37 ° C. for 2 hours, and the supernatant was collected to prepare a sample. The amount of cytokine before and after shaking was determined by standardizing the solid-liquid ratio of carrier: serum amount = 30 mg: 1 ml, and the removal rate was measured.

[Production Example 1]
(Nonwoven fabric)
Thirty-six sea-island composite fibers, each of which is made of a core-sheath composite, were obtained using the following components under the 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 90% by weight, polypropylene 10% by weight
Sea component: Copolymerized polyester composite ratio (weight ratio) containing ethylene terephthalate unit as a main repeating unit and 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component; core: sheath: sea = 44: 44: 12
A sheet-like material composed of 85% by weight of the fibers and 15% by weight of polypropylene having a diameter of 20 μm was produced, and then a needle punch was performed to obtain a nonwoven fabric. Next, this non-woven fabric is treated with an aqueous solution of sodium hydroxide at 90 ° C. to dissolve a copolymer polyester containing 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component, with the ethylene terephthalate unit of the sea component as the main repeating unit. Thus, a nonwoven fabric having a core-sheath fiber diameter of 5 μm and a bulk density of 0.05 g / cm ³ (total basis weight 250 g / m ² ) was produced (nonwoven fabric 1).

(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 above-mentioned raw yarn 1 was immersed in this, and left still 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 7.0 g of α-chloroacetamidomethylated polystyrene fiber (intermediate 1). The zeta potential was −21 mV.

(Adsorbent (adsorbent, adsorbent carrier))
5 g of intermediate A1 was immersed in a solution of 50 g of N, N-dimethyloctylamine and 8 g of potassium iodide in 360 mL of DMF and heated in a bath at 85 ° C. for 3 hours. The fiber was washed with isopropanol, then immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 8.3 g of dimethyloctylammoniumated fiber (adsorbent 1). The zeta potential was -1 mV.

  Further, 5 g of Intermediate 1 was immersed in a solution of 50 g of N, N-dimethylhexylamine and 8 g of potassium iodide in 360 mL of DMF, and heated in a 85 ° C. bath for 3 hours. The fiber was washed with isopropanol, dipped in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 9.3 g of dimethyllauryl ammoniumated fiber (adsorbent 2). The zeta potential was 1.2 mV.

[Example 1]
Heparin was collected from 50 ml of blood from healthy volunteers, and human natural IL-1 and IL-6 were dissolved therein so as to have a concentration of 500 pg / ml.

  150 mg each of adsorbent 1 and adsorbent 2 was packed in a column having an internal volume of 2 ml, and after circulating 25 ml of the blood at 37 ° C. for 1 hour, the composition of blood cells was examined with an automatic blood analyzer, and IL-1 The amount of IL-6 was quantified by the EIA method. The adsorption rate was calculated from the difference between before and after circulation. Adsorbent 1 had a lymphocyte adsorption rate of 19.5%, a granulocyte adsorption rate of 78%, and a monocyte adsorption rate of 85%, and an IL-1 adsorption rate and an IL-6 adsorption rate of 37% and 32%, respectively. It was. Similarly, in citrated plasma prepared using healthy volunteer blood, a decrease in the activity of coagulation factor XIII was found to be 12%. Adsorbent 2 had a lymphocyte adsorption rate of 21%, a granulocyte adsorption rate of 75%, and a monocyte adsorption rate of 83%, and an IL-1 adsorption rate and an IL-6 adsorption rate of 37% and 40%, respectively. Similarly, in citrated plasma prepared using healthy volunteer blood, a decrease in the activity of coagulation factor XIII was found to be 16%.

  Separately, as a result of the cytokine adsorption evaluation, the adsorbent 1 has an adsorption rate of IL-1 and IL-6 of 56% and 88%, respectively, and the adsorbent 2 has an adsorption rate of 74 and IL-1 and IL-6, respectively. %, 93%.

[Comparative Example 1]
The test similar to Example 1 was done using the intermediate body 1 (blood volume is 25 ml). Lymphocyte adsorption rate was 19.5%, granulocyte adsorption rate was 78%, monocyte adsorption rate was 78%, IL-1 adsorption rate and IL-6 adsorption rate were 2% and 3%, respectively. . Similarly, in citrated plasma prepared using healthy volunteer blood, a decrease in the activity of coagulation factor XIII was found to be 16%. Separately, as a result of cytokine adsorption evaluation, the adsorption rates were 12% and 13% for IL-1 and IL-6, respectively.

As is apparent from the results of Example 1 and Comparative Example 1 described above, the present invention, which is an adsorbent having a zeta potential of −20 mV or more, can adsorb granulocytes and monocytes in blood with high efficiency, and further cytokines. Can be adsorbed with high efficiency at the same time.
[Examples 2 to 7]
In order to obtain a correlation between the adsorption rate of granulocytes and monocytes and the fiber diameter, an adsorption test was performed according to the following procedure using blood (Ht = 43%) of a healthy human subject.

(Measurement of granulocyte / monocyte adsorption rate and correlation of fiber diameter)
Fibers with fiber diameters of 2 μm, 3 μm, 4 μm, 6 μm, 10 μm, and 17 μm were prepared, respectively, immersed in human blood so that the solid-liquid ratio was 20 mg / ml, maintained at 37 ° C., and 3 per minute. Invert mixing was performed for 5 minutes. After this, the fibers were removed, and the number of granulocytes (substitute the neutrophil values), monocytes, and lymphocytes in whole blood before immersion and in whole blood after immersion were respectively calculated by Sysmex's hemocytometer (XT1800iv). ) And the adsorption rate was measured. Table 1 shows the adsorption rate (removal rate) of each blood cell.

(Consideration of correlation between adsorption rate and fiber diameter)
In order to obtain a correlation between the adsorption rate of granulocytes and monocytes and the fiber diameter, an adsorption test was performed using blood (Ht = 43%) of healthy human subjects. In this examination, a removal rate of about ½ is obtained for circulation in the column. As a result, as is apparent from Table 1, the adsorption rate of lymphocytes is low and does not fluctuate with this range of fiber diameters, and the removal rate of granulocytes (neutrophils) and monocytes. Was found to be a region where the fiber diameter exceeded about 3 microns.
[Examples 8 to 10 and Comparative Examples 2 to 4]
In the following examples, the relationship between zeta potential and lipopolysaccharide adsorption ability and the effect of promoting production of interferon gamma were examined.

(Measurement of zeta potential)
The surface zeta potential was measured under the same conditions as in Example 1 above.

(Evaluation of cytokine adsorption)
Cytokine adsorption evaluation was carried out by adding human natural IL-1 and IL-6 to fetal calf serum and adjusting to 500 pg / ml. An adsorbent carrier was added to the serum, shaken at 37 ° C. for 2 hours, and the supernatant was collected to prepare a sample. The amount of cytokine before and after shaking was determined by standardizing the solid-liquid ratio of carrier: serum amount = 30 mg: 1 ml, and the removal rate was measured. The quantification was performed using the EIA method using a commercially available kit (IL-1: human IL-1β ELISA kit manufactured by R & D System, IL-6: manufactured by Kamakura Technoscience).

(Interferon gamma production evaluation)
The adsorbent-stimulated blood was obtained by passing it through a polypropylene cylindrical column having an internal volume of 2 ml and filled with 0.3 g of adsorbent using 10 ml of human volunteer blood at a blood flow rate of 2 ml / min. Lymphocyte fractions are separated by Ficoll density gradient centrifugation for blood that has passed through the column. The lymphocyte concentrate 8 hours after contact with the blood before contact with the adsorbent was stimulated with 1-10 μg of PHA (phytohemagglutinin-L: manufactured by Wako Pure Chemical Industries), and the concentration of interferon gamma before and after stimulation was measured. . Quantification was performed using a commercially available kit (human interferon gamma ELISA kit manufactured by Endogen) using the EIA method. (Interferon gamma concentration after stimulation / interferon gamma concentration before stimulation) was determined, and interferon production activity was determined.

(Measurement of blood cell count)
Quantification of the number of blood cells in the body fluid and measurement of the hematocrit value were performed using Sysmex XT-1800iV.

(Measurement of LPS)
The amount of LPS adsorbed was measured with a Toxinometer manufactured by Wako Pure Chemical Industries. Wako Pure Chemical Industries LPS (catalog number: 120-04531) is dispersed in 1% FCS-added physiological saline to a concentration of 10 ng / ml, and incubated with 300 mg of adsorbent for 4 hours at 37 ° C in a water bath. went. The amount of LPS remaining in the supernatant was measured and similarly measured with a toxinometer. The removal amount and removal rate were determined from the difference and ratio with LPS in the additive solution. The standard value is that the removal rate is 90% or more, and the adsorption amount is LPS adsorption capacity of 100 pg / mg or more.

[Production Example 2]
(Nonwoven fabric)
Thirty-six sea-island composite fibers, each of which is made of a core-sheath composite, were obtained using the following components under the 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 90% by weight, polypropylene 10% by weight
Sea component: Copolymerized polyester composite ratio (weight ratio) containing ethylene terephthalate unit as a main repeating unit and 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component; core: sheath: sea = 44: 44: 12
After producing a sheet-like material composed of 75% by weight of this fiber and 25% by weight of polypropylene having a diameter of 20 μm, a nonwoven fabric was obtained by needle punching. Next, this non-woven fabric is treated with an aqueous solution of sodium hydroxide at 90 ° C. to make the ethylene terephthalate unit of the sea component the main repeating unit, and by dissolving a copolymer polyester containing 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component A non-woven fabric having a core-sheath fiber diameter of 4.5 μm and a bulk density of 0.03 g / cm ³ (total basis weight 200 g / m ² ) was produced (non-woven fabric 1).

(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. Immediately after the temperature was raised to 20 ° C., 5 g of the raw yarn 1 was immersed 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 washed thoroughly with methanol, washed with water, and dried to obtain 7.0 g of α-chloroacetamidomethylated polystyrene fiber (intermediate 2). The zeta potential was −23 mV.

(Adsorbent)
5 g of the intermediate 2 was immersed in a solution of 50 g of N, N-dimethyloctylamine and 8 g of potassium iodide in 360 mL of methanol and heated in a bath at 50 ° C. for 3 hours. The fiber was washed with isopropanol, immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 8.1 g of dimethyloctylammonium-containing fiber (adsorbent 3). The zeta potential was −0.3 mV.

  Further, 5 g of intermediate 3 was immersed in a solution of 50 g of N, N-dimethylhexylamine and 8 g of potassium iodide in 360 mL of methanol, and heated in a bath at 50 ° C. for 3 hours. The fiber was washed with isopropanol, immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 7.3 g of dimethylhexylammonium-containing fiber (adsorbent 4). The zeta potential was 2.2 mV.

[Example 8]
Heparin was collected from 50 ml of blood from healthy volunteers, and human natural IL-1 and IL-6 were dissolved therein so as to have a concentration of 500 pg / ml.

  150 mg each of adsorbent 3 and adsorbent 4 was packed in a column having an internal volume of 2 ml, and after circulating 25 ml of the blood at 37 ° C. for 1 hour, the composition of the blood cells was determined with an automatic blood analyzer (XT-1800iV manufactured by Sysmex Corporation). Further, the amounts of IL-1 and IL-6 were quantified by the EIA method. In adsorbent A3, lymphocyte counts decreased by 14.5%, granulocytes decreased by 72%, monocytes decreased by 82%, and IL-1 and IL-6 decreased by 33% and 52%, respectively. In Adsorbent A4, 21% decrease in lymphocyte count, 75% decrease in granulocytes and 83% decrease in monocytes were observed, and IL-1 and IL-6 decreased by 37% and 40%, respectively.

  The LPS removal rate was 98% for adsorbent 3 and 97% for adsorbent 4.

  Separately, as a result of cytokine adsorption evaluation, the removal rate of IL-1 and IL-6 was 56% and 88% for adsorbent A3, and that of IL-1 and IL-6 was 74% for adsorbent 4 respectively. 93%.

[Example 9]
(Extracorporeal circulation treatment)
An extracorporeal circulation column was prepared by packing 0.3 g of the adsorbent 3 into a polypropylene cylindrical column having an inner diameter of 1 cm and an internal volume of 2 mL. 12-week-old WKAH: Hkm rat (male) subcutaneously on the back of 4-dimethylaminoazobenzene-induced liver cancer cell KDH-8 {Yano Satoshi, Hokkaido Medical Journal, Vol. 68, No. 654-664 (1993)} 1 × 10 ^Six were inoculated. The cancer cells were engrafted with a 100% probability. (Usually, the tumor grows from 1 week after inoculation and dies in 5.5 weeks after inoculation.) The extracorporeal circulation column is pre-washed with physiological saline containing 1000 units of heparin sodium before extracorporeal circulation, and an additional 500 mL It was used after washing with normal saline.

  Rats two weeks after KDH cell inoculation were treated with extracorporeal circulation. Blood was collected from the femoral artery and the number of granulocytes and lymphocytes before extracorporeal circulation was confirmed with an automatic blood analyzer. As a result, granulocytes were 9300 / μL and lymphocytes were 8100 / μL. After passing through the extracorporeal circulation column, a circuit for returning blood to the femoral vein was prepared, and the extracorporeal circulation was performed at a blood flow of 2 ml / min for 1 hour. During extracorporeal circulation, heparin sodium injection solution (manufactured by Ajinomoto Co., Inc.) was continuously infused at a rate of 200 U / h. The extracorporeal circulation column was pre-washed with physiological saline containing 1000 units of heparin sodium before extracorporeal circulation, and further washed with 500 mL of physiological saline. After extracorporeal circulation, treatment such as suturing was performed, blood was collected 160 hours later, and the number of granulocytes and lymphocytes was measured by an automatic blood analyzer. As a result, granulocytes were 6700 / μL, lymphocytes were 11400 / It was μL, and lymphocytes increased and granulocytes decreased compared to before treatment. The rats were further bred for 3 weeks and then extracorporeally circulated by the same procedure as 2 weeks after KDH cell inoculation. When the number of granulocytes and lymphocytes before extracorporeal circulation was confirmed by an automatic blood analyzer, granulocytes were 28000 / μL and lymphocytes were 7400 / μL. Blood was collected 160 hours after extracorporeal circulation, and the number of granulocytes and lymphocytes was measured by an automatic hematology analyzer. The number of granulocytes was 26700 / μL and that of lymphocytes was 8400 / μL. Spheres increased and granulocytes decreased.

[Comparative Example 2]
The same test as in Example 8 was performed using Intermediate 2 (blood volume was 25 ml). The number of lymphocytes decreased by 19.5%, granulocyte by 78% and monocyte by 78%. IL-1 and IL-6 decreased by 2% and 3%, respectively. The LPS removal rate was 78%. Separately, as a result of cytokine adsorption evaluation, the removal rates were 12% and 13% for IL-1 and IL-6, respectively.

[Comparative Example 3]
The intermediate body 2 was packed in a polypropylene cylindrical column of 0.3 g, an inner diameter of 1 cm, and an internal volume of 2 mL, and extracorporeal circulation was performed on rats two weeks after inoculation of KDH cells in the same manner as in Example 8. Blood was collected from the femoral artery and the number of granulocytes and lymphocytes before extracorporeal circulation was confirmed by an automatic blood analyzer. As a result, granulocytes were 10300 / μL and lymphocytes were 8400 / μL. Treatment such as suturing was performed, blood was collected after 160 hours, and the number of granulocytes and lymphocytes was measured by an automatic blood analyzer. The number of granulocytes was 15200 / μL, and that of lymphocytes was 8100 / μL. Compared to before, lymphocytes decreased and granulocytes increased.

[Example 10]
Interferon gamma production ability was evaluated using adsorbent 4 using human peripheral blood. When the adsorbent was not treated, the interferon gamma concentration ratio was 20.4 times. On the other hand, it became 35.2 times at the time of a process, and it turned out that immune activity is improving.

[Comparative Example 4]
Interferon gamma production ability was evaluated using intermediate 2 using human peripheral blood. When the adsorbent was not treated, the interferon gamma concentration ratio was 20.1 times. On the other hand, it became 21.2 times at the time of a process, and it turned out that an immune activity state does not change.

  From the results of Examples 8 to 10 and Comparative Examples 2 to 4 described above, the present invention, which is an adsorbent having a zeta potential of −20 mV or more, can adsorb granulocytes and monocytes in blood with high efficiency. Can be adsorbed with high efficiency at the same time. The changes in the number of granulocytes and lymphocytes after extracorporeal circulation treatment were found to change to a ratio considered normal, although the mechanism was unknown.

  According to the present invention, an adsorbent capable of efficiently adsorbing and removing leukocytes, inflammatory and immunosuppressive cytokines in blood and reducing the removal rate of useful components in blood is provided. Such an adsorbent of the present invention can be provided for various uses such as leukocyte removal therapy, immunostimulation therapy, and cancer treatment.

  This material is also suitable as an affinity chromatography column, therapeutic blood column, especially an extracorporeal circulation column in the form of petri dishes, bottles, membranes, fibers, hollow fibers, granular materials, or molded articles such as these. Can be used for

Claims (8)

Leukocyte removal consisting of a quaternary ammonium salt and / or a linear amino group bound to a water-insoluble carrier, with an adsorption rate of granulocytes in blood of 50% or more and an adsorption rate of monocytes of 50% or more. adsorbent for therapy. Adsorption rate of lymphocytes in the blood is 40% or less, according to claim 1 adsorbent according. The shape of the water-insoluble carrier, fiber, film, a shape that is selected from the group consisting of hollow fiber and beads, according to claim 1 or 2 adsorbent according. The shape of the water-insoluble carrier is in the form that the diameter of the projection of the fiber and the hollow fiber and the surface fiber diameter exceeds 3 [mu] m is selected from the group consisting of beads exceeding 3 [mu] m, according to claim 3 adsorbent according. The shape of the water-insoluble carrier is in the form of fiber diameter size of the projection of the fiber and the hollow fiber and the surface of 4-8 [mu] m is selected from the group consisting of beads of 4-8 [mu] m, the adsorption material according to claim 3, wherein . The shape of the water-insoluble carrier is in the form of fiber diameter size of the projection of the fiber and the hollow fiber and the surface of 4.5～8Myuemu is selected from the group consisting of beads of 4.5～8Myuemu, claim 3 The adsorbent described. The quaternary ammonium salts, N, N- dimethyl hexylamine, N, N- dimethyl octylamine and N, with N- dimethyl lauryl amine emissions or made salt compound selected from the group formed by bonding The adsorbent according to any one of claims 1 to 6 , wherein the linear amino group is a group formed by bonding tetraethylenepentamine . A blood treatment column for leukocyte removal therapy, wherein the adsorbent according to any one of claims 1 to 7 is filled in a container.