JP2007313288A - Adsorbing carrier and column for extracorporeal circulation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adsorbing material which can be suitably used for efficiently removing abnormally activated and proliferating leukocytes while suppressing the removal of useful leukocytes, and a blood treatment column using the adsorbing material. <P>SOLUTION: The leukocyte adsorbing material is characterized as follows: the ratio of adsorption of foxp3, CD4 and CD25-positive leukocytes containing water-insoluble carriers is 40% or less; and/or the ratio of adsorption of CD123-positive leukocytes is 60% or more. The shape of the adsorbing material is selected from among the shapes of a fiber, a membrane, a hollow fiber and a bead. The blood treatment column, the container of which is filled with the leukocyte adsorbing material, is suitable used for a leukocyte removing therapy. <P>COPYRIGHT: (C)2008,JPO&INPIT

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.

  Currently, diseases that are abnormally activated and caused by destruction or damage of self cells by proliferated leukocytes include rheumatoid arthritis, insulin-dependent diabetes, systemic lupus erythematosus (SLE), Sjogren's syndrome, scleroderma , Autoimmune diseases such as idiopathic thrombocytopenic purpura, Graves' disease / malignant anemia, Addison's disease, atrophic gastritis, hemolytic anemia, ulcerative colitis, myasthenia gravis, multiple sclerosis, hay fever, There are allergic diseases such as atopic dermatitis, allergic rhinitis, bronchial asthma, anaphylactic shock, etc. These treatments include steroids and other medications and blood cells that remove excess immune cells (white blood cells) from the blood Component removal therapy is being performed.

  In autoimmune diseases, proliferated leukocytes are involved in the disease, but for foxp3, CD4 and CD25 positive leukocytes (in the present invention, all of foxp3, CD4 and CD25 are positive). It is known to play an important role in the body's immune response. On the other hand, in allergic diseases, it is known that CD123-positive leukocytes (in the present invention, leukocytes in which CD123 is positive) are significantly proliferating compared to a healthy state, and CD123-positive The involvement of leukocytes in these diseases has been suggested.

Previous columns for removing leukocytes and granulocytes for the purpose of removing blood cell components (Patent Documents 1 and 2) are used to remove leukocytes themselves and granulocytes themselves in the carriers of these columns. Has been improved, but normal and abnormally activated leukocytes are also removed, resulting in insufficient normalization of the remaining cells.
JP-A-60-193468 JP-A-5-168706

  In view of the problems of the prior art, the present invention provides an adsorbent that can be suitably used to efficiently remove abnormally activated and proliferated leukocytes while suppressing the removal of useful leukocytes and blood using the same The purpose is to provide a processing column.

1. A leukocyte-adsorbing material for use in selectively adsorbing CD123-positive leukocytes from blood, comprising a water-insoluble carrier.
2. A leukocyte-adsorbing material comprising a water-insoluble carrier and having an adsorption rate of foxp3, CD4 and CD25-positive leukocytes of 40% or less and / or an adsorption rate of CD123-positive leukocytes of 60% or more.
3. 3. The leukocyte adsorbent according to 1 or 2 above, wherein the shape is selected from fibers, membranes, hollow fibers and beads.
4). 4. The leukocyte adsorbent according to any one of 1 to 3 above, comprising fibers or hollow fibers having a fiber diameter of 4 to 10 μm, and / or beads having a diameter of protrusions on the surface of the bead particles of 4 to 10 μm.
5). 5. The leukocyte adsorbent according to any one of 1 to 4, which is used for leukocyte removal therapy.
6). A blood treatment column obtained by filling a container with the leukocyte adsorbent according to any one of 1 to 5 above.

  The material of the present invention is useful for blood treatment and treatment of autoimmune diseases and allergic diseases by efficiently removing excessively activated and proliferated leukocytes such as granulocytes and monocytes that are unnecessary for the human body. .

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

  The leukocyte adsorbent in the present invention contains a water-insoluble carrier and is used for the purpose of selectively removing CD123-positive leukocytes from blood. “Selective removal of CD123-positive leukocytes from blood” means that when blood is brought into contact with an adsorbent, CD123-positive leukocytes are more adsorbed than lymphocytes that play an important role in the body's immune response. Preferably, the adsorption rate of CD123 positive leukocytes is higher than the overall leukocyte adsorption rate (adsorption rate as the number of all leukocytes including CD123 positive leukocytes). . The lymphocytes referred to in the present invention are used as a general term for so-called lymphocytes. According to the cell classification performed using XT-1800iV manufactured by Sysmex, B cells and T cells are not distinguished. On the other hand, although the leukocyte adsorbent of the present invention is also used for “non-selectively removing foxp3, CD4 and CD25 positive leukocytes”, this is compared with the adsorption rate of granulocytes involved in various diseases, It means that the leukocyte adsorption rate of all the foxp3, CD4 and CD25 positive leukocytes (adsorption rate as the number of all leukocytes including foxp3, CD4 and CD25 positive leukocytes) is removed at a lower adsorption rate, preferably This refers to removal of foxp3, CD4 and CD25 positive leukocytes at a lower adsorption rate compared to the typical leukocyte adsorption rate. That is, the leukocyte-adsorbing material of the present invention is “uses that require selective removal of CD123-positive leukocytes” or “uses that require non-selective removal of foxp3, CD4, and CD25-positive leukocytes” or these. It is used for both applications. Specific examples of such uses include therapeutic uses for autoimmune diseases and allergic diseases. Such specific autoimmune diseases include rheumatoid arthritis, insulin-dependent diabetes mellitus, systemic lupus erythematosus (SLE), Sjogren's syndrome, scleroderma, idiopathic thrombocytopenic purpura, Graves' disease / malignant anemia, Addison's disease, Examples include atrophic gastritis, hemolytic anemia, ulcerative colitis, myasthenia gravis, multiple sclerosis, and specific allergic diseases include hay fever, atopic dermatitis, allergic rhinitis, bronchi Examples include asthma and anaphylactic shock.

  The adsorption rate of foxp3, CD4 and CD25 positive leukocytes by the leukocyte adsorbent in the present invention is desirably 40% or less, and more preferably 30% or less. When the adsorption rate exceeds 40%, the balance of the immune response necessary for the human body is lost, and the disease caused by the destruction or damage of own cells by the proliferated leukocytes is aggravated.

  Here, the adsorption rate of foxp3, CD4 and CD25 positive leukocytes is the adsorption rate of leukocytes in which all of foxp3, CD4 and CD25 are positive, and means the total adsorption rate of all three types. is not.

  The adsorption rate of CD123 positive leukocytes by the leukocyte adsorbent in the present invention is desirably 60% or more, and more preferably 70% or more. If the adsorption rate is less than 60%, the expected therapeutic effect for various diseases cannot be obtained.

  Further, the adsorption rate in the present invention refers to blood cells before and after circulating 25 ml of blood in a 2 ml internal column packed with an adsorbent at 37 ° C. for 1 hour at a flow rate of 1.3 ml / min. The number is measured with a blood cell analyzer, calculated from the following formula, and the value after the decimal point is rounded off. The column size may be changed by calculating the blood volume and blood flow rate from the above ratio.

Adsorption rate (%) = [(number of blood cells in blood before column circulation) − (blood cell amount in blood after column circulation)] / (blood cell amount in blood before column circulation) × 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.

  As a basic configuration of such an adsorbent, a water-insoluble carrier is preferably used.

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. The water-insoluble carrier is composed of a water-insoluble material, but the water-insoluble material in the present invention means a material that is substantially or most of insoluble in water and is placed in water for 60 minutes. In this case, the weight loss is less than 2% of the initial weight. From the viewpoint of biocompatibility, olefinic compounds such as polypropylene and polyethylene are preferred, and polyamides and polyesters represented by polyethylene terephthalate are also preferred.
In the present invention, the shape of the water-insoluble carrier is not particularly limited, and examples thereof include various molded products such as powder, particles, fibers, and films. From the viewpoint of processability and pressure loss when a blood treatment column is used, the shape of fibers, membranes, hollow fibers or beads, or a combination of these shapes is preferred.

  In order to adsorb and remove abnormally activated leukocytes, the adsorption rate of CD123 positive leukocytes decreases when the diameter of the protrusions on the surface of these fibers, hollow fibers and bead particles used as a water-insoluble carrier exceeds 10 μm. Therefore, it is preferably 20 μm or less in practice, and more preferably 10 μm or less. Further, when the diameter is smaller than 4 μm, foxp3, CD4 and CD25 positive leukocytes are also removed by adsorption, so that the diameter is preferably 4 μm or more. However, the diameter of the fiber structure mixed for the purpose other than the removal of blood cells is not limited to this. In addition, the fiber diameter here is 10 samples of small pieces randomly taken from a non-woven fabric, photographed 1000 to 3000 times with a scanning electron microscope or the like, 10 from each sample, a total of 100 fibers. It means the one obtained by measuring the diameter and rounding off the first decimal place of the average value.

  In the present invention, in order to efficiently remove abnormally activated leukocytes, a functional group is immobilized on a water-insoluble carrier, or a water-insoluble carrier on which a functional group is immobilized is coated on a substrate. preferable. Among these, a quaternary ammonium salt, a linear amino group, or both of them (hereinafter referred to as a quaternary ammonium salt or the like) as a functional group is preferably immobilized on the water-insoluble carrier. Examples of reactive functional groups for immobilizing quaternary ammonium salts on water-insoluble carriers include active halogen groups such as halomethyl groups, haloacetyl groups, haloacetamidomethyl groups, and alkyl halide groups, epoxide groups, carboxyl groups, and isocyanates. An acid group, a thioisocyanic acid group, an acid anhydride group and the like can be mentioned. In particular, an active halogen group, especially a haloacetyl group, is easy to produce and has a moderately high reactivity, and is a quaternary ammonium salt. Such an immobilization reaction can be carried out under mild conditions, and the resulting covalent bond is chemically stable.

  As the functional group to be immobilized, a quaternary ammonium salt or the like is preferable, and each of these is a compound in which a tertiary amino group is chemically bonded to a polymer, ammonia, or a primary or secondary amino group. Means in a state chemically bound 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, those having 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. It is excellent from the viewpoint of adsorbing cytokine together with leukocytes. Such cytokines include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α. Examples include at least one cytokine selected from the group consisting of (TNF-α), transforming growth factor beta (TGF-β), angiogenic growth factor (VEGF), and immunosuppressive acidic protein (IAP). These are 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 to be applied. Specific examples of the compound having a tertiary amino group include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl- And hexylamine.

  Specific examples of the compound having such a linear amino group include diaminoethane, dipropylenetriamine, polyethyleneimine, N-methyl-2,2′-diaminodiethylamine, N-acetylethylenediamine, 1,2-bis (2 Alkylamines typified by -aminoethoxyethane) and the like are used, and ethyleneimines typified by diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and polyethyleneimine are preferably used. More preferably, ethyleneimines having 1 to 10 nitrogen atoms are used. In addition, about the linear amino group as used in the field of this invention, the thing of a structure with a partial branch is also included.

  The density per unit of the water-insoluble carrier of the quaternary ammonium salt or the like 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 exhibited, while it is too much. Then, the physical strength of the carrier after immobilization deteriorates and the function as an adsorbent tends to decrease, so the density is 0.01 to 2.0 mol, more preferably 0, per repeating unit of the water-insoluble carrier. .1 to 1.0 mol is preferable.

  As described above, the shape of the adsorbent is not particularly limited, but when used as a column, beads, fibers, membranes, hollow fibers are preferable, and the form is a knitted fabric, a woven fabric, a nonwoven fabric, etc. The fiber structure is 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 create It can also be used as a column. Operation such as immobilization or mixing may be performed before processing into the above-mentioned shape.

In the adsorbent of the present invention, the form is particularly preferably a non-woven fabric. In that case, if the bulk density of the nonwoven fabric is too large, it is likely to be clogged, and conversely if it is too small, the shape retainability deteriorates, and therefore it is preferably 0.02 to 0.15 g / cm ³ , more preferably 0. Those of 0.05 to 0.15 g / cm ³ are used.

  The nonwoven fabric used in the present invention may be produced from a single fiber, but is particularly preferably produced 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 needle punched to improve shape retention and to adjust the bulk density, and then the sea component is dissolved. Can be manufactured easily.

Preferred materials for the water-insoluble carrier are as described above. Specific examples of the quaternary ammonium salt and / or linear amino group polymer that can be used alone 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 - polysulfone type polymer represented by like, polyetherimide, polyimide, polyamide, polyether, polyphenylene sulfide, polystyrene, and the like acrylic polymers And what has a reactive functional group which can fix | immobilize an amino group by a covalent bond is used. 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.

  In the adsorbent of the present invention, a quaternary ammonium salt and / or a linear amino group is selected as a functional group, and a polymer in which this is immobilized is formed into a fiber, a hollow fiber and a bead as a water-insoluble carrier itself. In addition to manufacturing, it is preferable to manufacture fibers, hollow fibers and beads, particularly by coating a polymer having a quaternary ammonium salt and / or a linear amino group on a substrate such as a nonwoven fabric from the viewpoint of productivity. You can also. In this case, 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 these solutions, 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 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 is composed of a column filled with adsorbents formed in a flat plate shape, a cylindrical filter in which the adsorbent is wound into a cylindrical shape, and a cylindrical container having a blood inlet and a blood outlet at both ends. A column that is housed, and a hollow cylindrical filter in which an adsorbent is wound in a cylindrical shape, and is housed in a cylindrical container having a blood inlet and a blood outlet with both ends sealed. The column is preferably provided with a blood outlet provided at a site leading to the outer peripheral part of the hollow cylindrical filter, and a blood outlet of the container provided at a site leading to the inner peripheral part of the hollow cylindrical filter. Among them, the column using the cylindrical hollow filter is a method in which most of the activated 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 remaining without being removed. The activated leukocytes are also most preferable because the activated leukocytes can be efficiently removed so that the activated leukocytes can be sufficiently removed even by the non-woven fabric having a small area in the inner peripheral portion of the cylindrical filter.

Hereinafter, the present invention will be described more specifically with experimental examples, but the content of the invention is not limited to the examples. In addition, the fiber diameter collects 10 small-piece samples at random from the nonwoven fabric, takes 1000 to 3000 times photographs with a scanning electron microscope (manufactured by JEOL Ltd., JSM-5400LV), 10 from each sample, The diameters of a total of 100 fibers were measured and calculated by rounding to the first decimal place.
(Nonwoven fabric 1)
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: ethylene terephthalate unit as a main repeating unit, and copolymerized polyester composite ratio (weight ratio) containing 3% by weight of 5-sodiumsulfoisophthalic acid as a copolymer component; core: sheath: sea = 44: 44: 12
After producing a sheet-like material composed of 85% by weight of this fiber (diameter 30 μm) and 15% by weight of polypropylene having a diameter of 20 μm, a nonwoven fabric was obtained by needle punching (nonwoven fabric 1).
(Nonwoven fabric 2)
Next, this non-woven fabric 1 is treated with an aqueous solution of sodium hydroxide at 90 ° C. (3 wt%) to make a sea component “ethylene terephthalate unit as a main repeating unit and containing 3 wt% of 5-sodium sulfoisophthalic acid as a copolymer component. By dissolving the “copolyester”, 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 2).
(Intermediate 1)
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 2 was dipped 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 washed thoroughly with methanol, washed with water, and dried to obtain 7.0 g of α-chloroacetamidomethylated polystyrene fiber (intermediate 1).
(Adsorbent 1)
5 g of Intermediate 1 was immersed in a solution of 50 g of N, N-dimethyloctylamine and 8 g of potassium iodide in 360 mL of dimethyl sulfoxide (DMF) and heated in a 85 ° C. bath for 3 hours. The heated fiber was washed with isopropanol, immersed in 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 8.3 g of dimethyloctylammoniumated fiber (adsorbent 1).
(Adsorbent 2)
In a solution of 0.7 g of tetraethylenepentamine and 6.4 g of triethylamine in 360 mL of dimethyl sulfoxide, 5 g of intermediate 1 was heated in a 30 ° C. bath for 3 hours. The heated fiber was washed with methanol and then with water to obtain a tetraethylenepentaminated fiber (adsorbent 2).
[Example 1]
Heparin blood was collected from 50 ml of healthy volunteer's blood, and the following examination was performed.

  150 mg of the adsorbent 1 was sampled and packed in a column with an internal volume of 2 ml. After circulating 25 ml of the blood at 37 ° C. for 1 hour at a flow rate of 1.3 ml / min, the composition of blood cells was examined with an automatic blood analyzer. The adsorption rate was calculated from the following formula, and the value was calculated by rounding off the numerical value after the decimal point.

Adsorption rate (%) = [(number of blood cells in blood before column circulation) − (number of blood cells in blood after column circulation)] / (number of blood cells in blood before column circulation) × 100
PE anti-human foxp3 Staining Kit (manufactured by BAY BIOSCCIENCE Co., Ltd.), FITC anti-human CD4 (manufactured by BAY BIOSCCIENCE Co., Ltd.), phycoerythrin-Cy5 EN-BAY C Using foxp3, CD4 and CD25 for fluorescent labeling, FACS hemolysis reagent (manufactured by Becton Dickinson Co., Ltd.) was added and reacted for 30 minutes in the dark. After hemolysis, centrifugation and phosphate buffer were performed. Blood cells were washed with physiological saline. Thereafter, foxp3, CD4 and CD25 positive leukocytes were examined using a flow cytometer (FACSCalibur, manufactured by Becton Dickinson Co., Ltd.). Moreover, after fluorescently labeling CD123 using PE anti-human CD123 (manufactured by BAY BIOSCCIENCE Co., Ltd.), FACS hemolysis reagent (manufactured by Becton Dickinson Co., Ltd.) was added and allowed to react in the dark for 30 minutes to carry out hemolysis treatment. Later, the cells were centrifuged and the blood cells were washed with phosphate buffered saline. Thereafter, CD123 positive leukocytes were examined using a flow cytometer. Lymphocyte adsorption rate was 20%, granulocyte adsorption rate was 78%, monocyte adsorption rate was 78%, foxp3 and CD4 and CD25 positive leukocyte adsorption rate was 25%, and CD123 positive leukocyte adsorption rate was 71%. The results are shown in Table 1.

[Example 2]
The same experiment as in Example 1 was performed using the adsorbent 2.
As a result, the lymphocyte adsorption rate was 6%, granulocyte adsorption rate 51%, monocyte 50% adsorption rate, foxp3 and CD4 and CD25 positive leukocyte adsorption rate 23%, and CD123 positive leukocyte adsorption rate 61%. The results are shown in Table 1.

[Example 3]
Using the nonwoven fabric 2, the same experiment as in Example 1 was performed.
As a result, lymphocyte adsorption rate was 20%, granulocyte adsorption rate was 60%, monocyte adsorption rate was 72%, foxp3 and CD4 and CD25 positive leukocyte adsorption rate was 34%, and CD123 positive leukocyte adsorption rate was 70%. The results are shown in Table 1.
[Comparative Example 1]
Using the nonwoven fabric 1, the same test as in Example 1 was performed (blood volume is 25 ml). The adsorption rate was 9% for lymphocytes, 10% for granulocytes, 12% for monocytes, 11% for foxp3 and CD4 and CD25 positive leukocytes, and 11% for CD123 positive leukocytes. The results are shown in Table 1.

Claims (6)

A leukocyte-adsorbing material for use in selectively adsorbing CD123-positive leukocytes from blood, comprising a water-insoluble carrier. A leukocyte-adsorbing material comprising a water-insoluble carrier and having an adsorption rate of foxp3, CD4 and CD25-positive leukocytes of 40% or less and / or an adsorption rate of CD123-positive leukocytes of 60% or more. The leukocyte adsorbent according to claim 1 or 2, wherein the shape is selected from fibers, membranes, hollow fibers and beads. The leukocyte adsorbent according to any one of claims 1 to 3, comprising fibers or hollow fibers having a fiber diameter of 4 to 10 µm, and / or beads having a diameter of protrusions on the surface of the bead particles of 4 to 10 µm. . The leukocyte adsorbent according to any one of claims 1 to 4, which is used for leukocyte removal therapy. A blood treatment column comprising a container filled with the leukocyte adsorbent according to claim 1.