JPWO2006049163A1 - Body fluid treatment material that generates bradykinin capable of whole blood treatment - Google Patents

Abstract

An object of the present invention is to provide a bodily fluid treatment material for obtaining a bodily fluid in which bradykinin is appropriately generated directly in whole blood without losing useful substances such as albumin as much as possible. It is to provide a column to be made. The present invention is a body fluid treatment material obtained by immobilizing a tryptophan derivative and a polyanionic compound on a water-insoluble porous carrier, and the amount of polyanionic compound immobilized is 0.10 μmol or more per 1 ml of body fluid treatment material with a wet volume. The body fluid treatment material is 5 μmol or less, and the molar ratio of the amount of immobilized tryptophan derivative and the amount of immobilized polyanionic compound is 1 or more and 70 or less. By using the body fluid treatment material of the present invention, an appropriate amount of bradykinin can be safely generated by whole blood treatment.

Description

  The present invention relates to a body fluid treatment material for obtaining a body fluid containing moderately bradykinin, a method for generating bradykinin in a body fluid using the body fluid treatment material, and moderately bradykinin in the body fluid using the body fluid treatment material. In particular, the present invention relates to a body fluid treatment material capable of processing whole blood.

  Bradykinin is a peptide having a physiological activity consisting of 9 amino acids discovered by Rochae Silva in 1949. Bradykinin is released by the action of kallikrein on the high molecular weight kininogen, and the released bradykinin is degraded by kininase II (identical to angiotensin I converting enzyme).

  The mechanism of bradykinin production is considered as follows. That is, factor XII of the blood coagulation system is activated to become factor XIIa. This factor XIIa converts prekallikrein in blood into kallikrein. Furthermore, this kallikrein acts on high molecular kininogen in the blood to release bradykinin. On the other hand, kallikrein appearing in this process has the property of activating factor XII.

  In particular, the increase in blood bradykinin concentration is caused by the fact that factor XII is activated by contact with a foreign substance surface, and glass, celite, kaolin, collagen, etc. have been known for a long time as substances to be activated. In addition, it is known to be activated by stearic acid, ellagic acid, sulfatide, plasmin, kallikrein and the like. According to Patent Document 1, it is described that factor XII is activated by contact with the surface of many negatively charged medical materials, and the bradykinin concentration increases accordingly.

  Bradykinin has a strong vascular smooth muscle relaxing action and has the function of dilating blood vessels and lowering blood pressure. This is because bradykinin binds to the bradykinin 2 receptor of vascular endothelial cells and contracts vascular endothelial cells, increasing capillary permeability, and at that time, nitric oxide is produced and arteriole smooth muscle is relaxed It is thought that the blood vessels dilate.

  Hypertension, which is a typical lifestyle-related disease, is most likely to trigger stroke, with a high risk of developing physical disability, fatal left ventricular failure, myocardial infarction, cerebral hemorrhage, cerebral infarction, and renal failure at an early age. It is a risk factor. Angiotensin converting enzyme inhibitors are generally used as antihypertensive drugs for these hypertensive patients. In addition to their angiotensin converting enzyme inhibitory activity, angiotensin converting enzyme inhibitors include bradykinin, which produces vasodilator nitric oxide in endothelial cells. It also suppresses kininase II activity to be hydrolyzed, and exhibits a hypotensive action by delaying the degradation of bradykinin.

  Furthermore, in recent years, the number of patients who develop arteriosclerosis is increasing with the westernization and aging of eating habits, and arteriosclerosis is closely related to hypertension. When high pressure is constantly applied to the artery due to high blood pressure, a wound is generated in the artery wall, and fat in the blood penetrates from the wound to cause arteriosclerosis. In addition, as arteriosclerosis progresses, blood vessels become narrower and blood flow deteriorates, and in order to improve it, the heart strengthens its pulsation and raises the blood pressure.

  Among arteriosclerosis, a disease causing blockage of peripheral blood vessels is called obstructive arteriosclerosis. In this disease, peripheral blood vessels become narrower and occluded, resulting in poor peripheral blood circulation. Symptoms such as sensation of limbs, numbness, intermittent claudication, resting pain, ulcers, gangrene, etc., and limb amputation To. For this reason, blood flow is improved by the action of bradykinin having a vasodilatory effect, and it is expected to improve these symptoms.

  On the other hand, when the blood concentration becomes high due to the powerful vasodilatory effect of bradykinin, anaphylaxis-like symptoms such as rapid blood pressure reduction and nausea may be caused. In particular, blood purification therapy by extracorporeal circulation, which has been actively performed in recent years, is a treatment for removing a pathogenic substance from blood, and at that time, bradykinin is excessively caused by contact of certain medical materials with blood / plasma. It is a problem that can cause anaphylaxis-like symptoms. According to Patent Document 1, the above symptoms appear when the bradykinin concentration in plasma becomes 4000 pg / ml or more.

  In order to solve these problems, Patent Document 1 and Patent Document 2 describe an adsorbent using hydrophobicity for the purpose of removing bradykinin from blood, but both are intended for adsorption removal of bradykinin. It is.

As described above, in the conventional technique, there is only a method of generating bradykinin randomly in the blood in whole blood processing or removing bradykinin, and lowering blood pressure by vasodilatory action without causing anaphylactoid symptoms, Alternatively, it has been desired to develop a whole blood processing method for generating bradykinin to the extent that blood flow is improved for patients with obstructive arteriosclerosis.
JP-A-6-296861 JP-A-6-296864

  In view of the present situation as described above, the present invention provides a body fluid treatment material characterized by generating bradykinin in a body fluid to such an extent that blood pressure can be reduced without significant side effects or blood flow can be improved, and the body fluid treatment material. Is a method for generating bradykinin in body fluids using this, and a column for generating bradykinin using this body fluid treatment material, which minimizes the loss of blood cells with minimal loss of useful substances such as albumin. In addition, the present invention provides a body fluid treatment material and a column, which are capable of treating whole blood.

  The inventors of the present invention have conducted extensive research on a body fluid treatment material capable of processing whole blood without losing useful substances such as albumin as much as possible and generating a controlled amount of bradykinin in body fluids, particularly blood. As a result, a body fluid treatment material in which a tryptophan derivative and a polyanionic compound are immobilized on a water-insoluble porous carrier, the polyanionic compound is immobilized in a certain amount, and the immobilized amount of the tryptophan derivative and the polyanionic compound are immobilized. A body fluid treatment material with a specific molar ratio is a body fluid treatment material that moderately generates bradykinin in body fluids, especially blood, and safely treats whole blood by minimizing the loss of blood cells. As a result, the present invention has been completed.

  That is, the first invention of the present invention is a body fluid treatment material obtained by immobilizing a tryptophan derivative and a polyanionic compound on a water-insoluble porous carrier, and the amount of polyanionic compound immobilized per 1 ml of body fluid treatment material having a wet volume. Whole blood treatment is possible in which the molar ratio of the amount of immobilized tryptophan derivative and amount of immobilized polyanionic compound per ml of body fluid treatment material of 0.10 μmol to 1.5 μmol is 1 to 70 The present invention relates to a body fluid treatment material that generates bradykinin. The second invention relates to a method for generating bradykinin, wherein the body fluid treatment material is brought into contact with body fluid. The third invention is characterized in that a body fluid treatment material for generating bradykinin is filled in a container having an inlet and an outlet for the liquid and provided with means for preventing the body fluid treatment material from flowing out of the container. The present invention relates to a column that generates bradykinin capable of processing whole blood.

According to the present invention, it is possible to obtain a body fluid in which bradykinin is appropriately generated directly in a body fluid, particularly whole blood, without losing useful substances such as albumin as much as possible. INDUSTRIAL APPLICABILITY The present invention is effective as a method for lowering blood pressure in hypertensive patients or improving blood flow in patients with arteriosclerosis, particularly obstructive arteriosclerosis.

It is a schematic sectional drawing of one Example of the column which generate | occur | produces the bradykinin of this invention. It is a graph which shows the result of having investigated the relationship between the flow velocity and pressure loss using three types of gels.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Body fluid inlet 2 Body fluid outlet 3 Body fluid processing material which generates bradykinin 4, 5 mesh (body fluid processing material outflow prevention means)
6 Column 7 Column that generates bradykinin

  The body fluid in the present invention refers to blood, plasma, or serum.

  The polyanionic compound in the present invention refers to a compound having a plurality of anionic functional groups in the molecule. The anionic functional group in the present invention refers to a functional group that has a neutral pH and is negatively charged, such as a carboxyl group, a sulfonic acid group, a sulfate ester group, and a phosphate ester group. Among these, a carboxyl group, a sulfonic acid group, and a sulfate ester group are preferable from the viewpoint of the ability to generate bradykinin appropriately, and a sulfate ester group is particularly preferable from the viewpoint of excellent ability to control the generation amount.

  Representative examples of such polyanionic compounds include polyacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, and styrene-maleic acid copolymer. Compounds, synthetic acidic polysaccharides such as dextran sulfate and carboxymethylcellulose, biological mucopolysaccharides having sulfate ester groups such as chondroitin sulfate, dermatan sulfate and ketalan sulfate, N-sulfonic acid groups such as heparin and heparan sulfate, and sulfuric acid Examples include acidic mucopolysaccharides having ester groups, polysaccharides having biological anionic functional groups such as chondroitin and phosphomannan, and biological nucleic acids such as deoxyribonucleic acid and ribonucleic acid. Limited But it is not.

  Among these typified compounds, synthetic compounds can be used rather than using biologically derived compounds as they are because they can obtain highly pure substances at low cost and control the amount of anionic functional group introduced. It is more practical to use. From these points, synthetic polyanionic compounds such as polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, styrene-maleic acid copolymer, and dextran Synthetic acidic polysaccharides such as sulfuric acid and carboxymethyl cellulose are preferably used. Among them, polyacrylic acid, polystyrene sulfonic acid, and dextran sulfate are preferable from the viewpoint of inexpensiveness, and dextran sulfate is particularly preferable from the viewpoint of safety.

  The molecular weight of the polyanionic compound is preferably 1000 or more, particularly 3000 or more, from the viewpoint of generating bradykinin. The upper limit of the molecular weight of the polyanionic compound is not particularly limited, but is preferably 1,000,000 or less from the practical viewpoint.

  In the present invention, there are various methods for immobilizing a polyanionic compound on a water-insoluble porous carrier, and any method may be used. As a typical method, (1) a polyanionic compound is used by using radiation or an electron beam. There are a method of covalently bonding to the surface of a water-insoluble porous carrier by a graft method, and a method of (2) covalently bonding a polyanionic compound by a chemical method via a functional group of the water-insoluble porous carrier.

  Among these, considering that the present invention is a body fluid treatment material in which a polyanionic compound and a tryptophan derivative are immobilized, a method of chemically bonding a polyanionic compound via a functional group is a method of tryptophan derivative. Since the immobilization can be performed by the same method, it can be said to be a simpler and preferable method in the present invention.

  The tryptophan derivative in the present invention refers to a compound having a structure similar to tryptophan having tryptophan, tryptophan ethyl ester, tryptophan esters such as tryptophan methyl ester, and indole ring such as tryptamine and tryptophanol. Further, these tryptophan derivatives may be any of L-form, D-form, DL-form, and mixtures thereof. Further, it may be a mixture of two or more tryptophan derivatives. Among these tryptophan derivatives, tryptophan is preferred for safety. Among them, L-tryptophan is a natural amino acid, its safety data is abundant, and it is the cheapest and easy to obtain. Most preferably used.

  As a method for immobilizing a tryptophan derivative in the present invention, a method in which a tryptophan derivative is covalently bonded by a chemical method via a functional group of a water-insoluble porous carrier is preferably used.

  The immobilization amount of the polyanionic compound in the present invention is 0.10 μmol or more and 1.5 μmol or less per 1 ml of the wet body fluid treatment material, and the molar ratio of the immobilization amount of the tryptophan derivative and the polyanionic compound immobilization amount is 1 or more. It must be 70 or less.

The tryptophan derivative immobilization amount and the polyanionic compound immobilization amount molar ratio (TR / PA ratio) in the present invention are values calculated by the following equation.
TR / PA ratio = number of moles of immobilized tryptophan derivative per 1 ml of body fluid treatment material with a wet volume / number of moles of polyanion immobilized with 1 ml of body fluid treatment material of a wet volume. As a result of intensive studies on the amount of tryptophan derivative immobilized and blood cell passage when whole blood was directly processed, surprisingly, the amount of polyanionic compound immobilized was 0.10 μmol or more per 1 ml of body fluid treatment material in a wet volume. By controlling the TR / PA ratio to 1 to 70 and not more than 1.5 μmol, it has been found that leukocytes and platelets have good permeability while generating bradykinin at a level that is unlikely to cause anaphylactoid symptoms. It was.

  In the present invention, the immobilization amount of the polyanionic compound is 0.10 μmol or more and 1.5 μmol or less per 1 ml (wet volume) of the body fluid treatment material. When this amount of immobilization is less than 0.10 μmol, the generation of bradykinin is small and the permeability of leukocytes and platelets deteriorates. When whole blood perfusion treatment is performed, the number of leukocytes in pooled blood decreases. On the other hand, when the amount is more than 1.5 μmol, a large amount of bradykinin is generated even if the tryptophan derivative is immobilized. The amount of immobilization of the polyanionic compound is preferably 0.12 μmol or more and 1.0 μmol or less, and most preferably 0.15 μmol or more and 0.50 μmol or less from the viewpoint that a moderate amount of bradykinin generation and good blood cell passage can be exhibited. is there.

  In the present invention, the molar ratio (TR / PA ratio) between the immobilized amount of tryptophan derivative and the immobilized amount of polyanionic compound is 1 or more and 70 or less. When the TR / PA ratio is less than 1, a large amount of bradykinin is generated. Conversely, when the TR / PA ratio is greater than 70, bradykinin is hardly generated. In addition, the permeability of leukocytes and platelets gradually deteriorates, and the number of leukocytes in the pooled blood decreases when whole blood perfusion treatment is performed. It is preferably 5 or more and 60 or less, and most preferably 10 or more and 50 or less, from the viewpoint that appropriate generation of bradykinin and good blood cell permeability can be exhibited.

  The volume of the wet body fluid treatment material in the present invention is determined as follows. That is, the body fluid treatment material is transferred to a measuring container such as a graduated cylinder as a slurry immersed in water, and the slurry-like body fluid treatment material in the measuring container is naturally settled. After this, a rubber mat or the like is laid so that the measuring container is not broken, and the measuring container is lightened about 5 to 10 times in the vertical direction from a height of about 5 to 10 cm (the body fluid treatment material once settled is extremely Add vibration by striking it (to the extent that it does not fly up). After standing for 15 minutes or more, the sediment volume of the body fluid treatment material is read. This vibration and stationary operation are repeated, and the stage where the sedimentation volume of the body fluid treatment material does not change is defined as the sedimentation volume of the body fluid treatment material in a wet state.

  As a method for measuring the amount of immobilized polyanionic compound in the present invention, a method for quantifying the content of an element contained in a polyanionic compound in a body fluid treatment material (for example, in the case of dextran sulfate, the sulfur content of a body fluid treatment material is determined. Quantification), a method of measuring the amount of dye decreased in a solution by contacting a dye solution having a property of binding to a polyanionic compound and a body fluid treatment material. Among them, the method using a dye solution is simple and The immobilization amount of the polyanionic compound can be accurately measured. Specifically, as shown in Example 1, when the polyanionic compound is dextran sulfate, polyacrylic acid, or the like, the fact that the compound has a property of binding to toluidine blue is used. The amount of immobilization of toluidine blue when the blue solution and the body fluid treatment material are brought into contact with each other can be measured very simply.

  The amount of the tryptophan derivative immobilized in the present invention can be quantified by utilizing the property of coloring when an aldehyde such as p-dimethylbenzaldehyde is condensed with the indole ring present in the molecule of the tryptophan derivative under strongly acidic conditions. Yes, edited by Koichi Yamada, Amino Acid Fermentation (Part 2), 43-45, Kyoritsu Shuppan, 1972). In addition, a method in which the indole ring existing in the molecule of the tryptophan derivative is quantified by utilizing the property of emitting fluorescence having a maximum near 350 nm when excited by light around 280 nm, or a compound in which the carrier itself does not contain nitrogen In this case, as shown in a specific method in Example 1, it can also be measured by quantifying the nitrogen content in the body fluid treatment material.

  The water-insoluble porous carrier in the present invention is solid at room temperature and normal pressure, is insoluble in water, and has pores of an appropriate size, that is, has a porous structure. As the shape of the water-insoluble porous carrier, any of a spherical shape, a granular shape, a flat membrane shape, a fibrous shape, a hollow fiber shape and the like can be used effectively, but a spherical shape or a granular shape is more preferably used because of easy handling.

  When the water-insoluble porous carrier is spherical or granular, the average particle diameter of the carrier is better as long as the body fluid treatment material of the present invention can be treated with whole blood, but in terms of bradykinin generation, Since the contact area with the treatment material increases, the smaller the average particle size, the better. As the bodily fluid treatment material in the present invention, the average particle size of the bodily fluid treatment material capable of generating whole blood treatment and appropriate bradykinin is preferably 100 μm or more and 1000 μm or less, from the viewpoint that good blood cell permeability and generation of bradykinin can be controlled. More preferably, it is 200 micrometers or more and 800 micrometers or less, Most preferably, they are 400 micrometers or more and 600 micrometers or less.

The exclusion molecular weight of the globular protein of the water-insoluble porous carrier is preferably 5 × 10 ⁵ or more. Exclusion limit molecular weight, as described in the book (Size Exclusion Chromatography, written by Sadao Mori, Kyoritsu Shuppan), penetrates into the pores when samples with various molecular weights flow in Size Exclusion Chromatography. The molecular weight of the smallest molecular weight that cannot be excluded (excluded). When the exclusion limit molecular weight of globular protein is less than 5 × 10 ⁵ , the amount of bradykinin generated increases and it is not practical. On the other hand, when the exclusion limit molecular weight of the globular protein exceeds 1 × 10 ⁸ , the pore size becomes too large and the surface area decreases, resulting in a decrease in the amount of bradykinin generated. From the above, the exclusion molecular weight of the globular protein of the water-insoluble porous carrier in the present invention is preferably 5 × 10 ⁵ or more and 1 × 10 ⁸ or less, more preferably 1 × 10 from the viewpoint of controlling the amount of bradykinin generated. ^{It is 6} or more and 1 × 10 ⁸ or less, more preferably 2 × 10 ⁶ or more and 1 × 10 ⁸ or less.

  The water-insoluble porous carrier in the present invention preferably has a functional group that can be used for binding in order to immobilize the polyanionic compound and the tryptophan derivative. Representative examples of such functional groups include amino groups, amide groups, carboxyl groups, acid anhydride groups, succinimide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, silanol groups, and tresyl groups. However, it is not limited to these. The water-insoluble porous carrier may be activated by a method such as a halogenated cyanide method, an epichlorohydrin method, a bisepoxide method, or a bromoacetyl bromide method. In view of the above, the epichlorohydrin method is particularly preferably used.

As the strength of the water-insoluble porous carrier of the present invention, those which are too soft and easily broken are not preferred. When fluid is flowed, if consolidation occurs, sufficient fluid flow will not be obtained, and the treatment time may be extended and the treatment may not be continued. It is preferable that it has a mechanical strength (hard). As used herein, the term “hard” means that the relationship between the pressure loss and the flow rate when the body fluid treatment material is uniformly packed in a cylindrical column and the aqueous liquid is flowed is at least 0.3 kgf / cm ² , as shown in the reference examples described later. The one that has a linear relationship.

  The material of the water-insoluble porous carrier in the present invention is not particularly limited, but is an organic carrier made of polysaccharides such as cellulose, cellulose acetate, dextrin, polystyrene, styrene-divinylbenzene copolymer, polyacrylamide, polyacrylic acid, polymethacrylic. Synthetic polymers such as acid, polyacrylic acid ester, polymethacrylic acid ester, and polyvinyl alcohol are representative examples. These have a coating layer such as a polymer material having a hydroxy group such as hydroxyethyl methacrylate, or a graft copolymer such as a copolymer of a monomer having a polyethylene oxide chain and another polymerizable monomer. You may do it. Among these, a synthetic polymer made of cellulose, polyvinyl alcohol, or the like is preferably used practically because it easily introduces an active group to the surface of the carrier.

  Of these, a carrier made of cellulose is most preferably used. The carrier made of cellulose (1) has relatively high mechanical strength and is tough, so it is less likely to be broken or produce fine particles, and when packed in a column, it is difficult to consolidate even if a body fluid flows at a high flow rate. Therefore, it is possible to flow a body fluid at a high flow rate, and (2) it has excellent points such as higher safety than a synthetic polymer carrier, and is most suitable as a water-insoluble porous carrier in the present invention. Can be used.

  Anticoagulants for extracorporeal circulation treatment using the column of the present invention include heparin, low molecular weight heparin, nafamostat mesylate, gabexate mesylate, argatroban, sodium citrate solution, acid citrate dextrose solution (ACD solution) Any citric acid-containing anticoagulant such as citrate phosphate phosphate dextrose solution (CPD solution) may be used. Among them, citrate-containing anticoagulant, heparin, low molecular weight heparin, and nafamostat mesylate can be cited as particularly preferred anticoagulants from the viewpoint of whole blood treatment.

  There are various methods for generating bradykinin in body fluids using the body fluid treatment material of the present invention. As a representative method, a body fluid is taken out and stored in a bag or the like, mixed with a body fluid treatment material to generate bradykinin, and then filtered to remove the body fluid treatment material to obtain a body fluid in which bradykinin is generated, body fluid There is a method of producing a column in which a body fluid treatment material is filled in a container equipped with a filter attached to the exit port, which has a flow inlet and an outlet, and through which a body fluid passes but a body fluid treatment material does not pass. . Either method may be used, but the latter method is easy to operate, and by incorporating it into an extracorporeal circuit, bradykinin can be efficiently supplied to a patient's body fluid online. It is most preferable as a method for generating bradykinin using a body fluid treatment material.

  The column of the present invention has an inflow port and an outflow port for body fluid, and is formed by filling the body fluid treatment material of the present invention in a container equipped with a filter that passes the body fluid but does not pass the body fluid treatment material. The capacity of the column of the present invention needs to be 100 ml or more from the viewpoint of generating moderate bradykinin. There is no limit on the volume of the column, but if the volume is too large, too much bradykinin is generated, and if too much blood is taken out of the body, there is a risk of lowering blood pressure. 1000 ml, more preferably 800 ml or less, even if incorporated in other blood purification therapy circuits such as hemodialysis, the extracorporeal circulation of blood does not become extremely large, and may occur as blood goes out of the body From the viewpoint of preventing a possible decrease in blood pressure as much as possible, it is most preferably 400 ml or less.

  Next, the column of the present invention will be described with reference to FIG. 1 which is a schematic sectional view of one embodiment.

  In FIG. 1, 1 is a body fluid inlet, 2 is a body fluid outlet, 3 is a body fluid treatment material that generates bradykinin appropriately, 4 and 5 are meshes, 6 is a column, and 7 is a column that generates bradykinin. However, the column for generating bradykinin in the present invention is not limited to such a specific example, and is a container having an inlet and an outlet for liquid and means for preventing outflow of body fluid treatment material out of the container. The shape is not particularly limited as long as it is filled with a body fluid treatment material that appropriately generates bradykinin.

  Hereinafter, the method of the present invention will be specifically described based on examples.

(Reference example)
Agarose material (Biogel A-5m manufactured by Bio-rad, particle size 50-100 mesh) on a glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with a filter with a pore size of 15 μm at both ends, vinyl-based high Molecular materials (Toyopearl HW-65 manufactured by Tosoh Corporation, particle size 50-100 μm) and cellulose materials (Cellulofine GC-700 m, particle diameter 45-105 μm manufactured by Chisso Corporation) were uniformly filled, respectively. Water was passed by a static pump, and the relationship between the flow rate and the pressure loss ΔP was determined. The result is shown in FIG.

As shown in FIG. 2, the flow rate of Toyopearl HW-65 and Cellulofine GC-700m increases in proportion to the increase in pressure, whereas Biogel A-5m causes compaction. It turns out that it does not increase. In the present invention, as in the former case, a material in which the relationship between the pressure loss ΔP and the flow rate has a linear relationship up to 0.3 kgf / cm ² is called hard.

(Example 1)
Water (85 ml), 4N NaOH aqueous solution (76 ml) and epichlorohydrin (68 ml) were added to 235 ml of porous cellulose beads having an average particle size of about 450 μm and a globular protein exclusion limit molecular weight of 5 × 10 ⁷ , and the mixture was allowed to react at 40 ° C. for 2 hours with stirring. It was. After the reaction, the beads were sufficiently washed with water to obtain epoxidized cellulose beads. The amount of epoxy groups of the epoxidized cellulose beads was 19.9 μmol / ml (wet volume).

  A dextran sulfate aqueous solution in which 35.4 g of dextran sulfate (sulfur content: about 18%, molecular weight: about 4000) was dissolved in 100 ml of water was prepared, 220 ml of epoxidized cellulose beads in a wet state were added, and the mixture was made alkaline with an aqueous NaOH solution. Then, it was made to react at 45 degreeC for 3 hours. After the reaction, the beads were sufficiently washed with water and brine, and then a solution prepared by dissolving 3.68 g of L-tryptophan in 120 ml of dilute aqueous NaOH was added and reacted at 55 ° C. for 6 hours. Thereafter, the beads were sufficiently washed with water and saline to obtain dextran sulfate and tryptophan-immobilized cellulose beads (A).

  This A was taken in a 0.2 ml test tube, and 3 ml of healthy human blood anticoagulated by adding 5 units of heparin to 1 ml of blood was added and shaken at 37 ° C. for 10 minutes. The bradykinin concentration in the supernatant was measured by radioimmunoassay and found to be 291 pg / ml.

  In addition, an acrylic column (volume 2.7 ml) with an inner diameter of 10 mm and a length of 34 mm, fitted with a 150 μm mesh polyethylene terephthalate mesh at both ends, is filled with A, and 5 units of heparin is added to 1 ml of blood for anticoagulation. 40 ml of healthy human blood was circulated at a flow rate of 6.5 ml / min for 2 hours. The number of blood cells in the pooled blood before and after 2-hour circulation was as shown in Table 2, and all blood cells showed good permeability.

  The amount of tryptophan immobilized on A was determined from the nitrogen content of the body fluid treatment material. That is, 1 ml of A was thoroughly washed with water, dried under reduced pressure at 60 ° C. for 6 hours or more, and then quantified with a trace total nitrogen analyzer. As shown in Table 1, the amount of A immobilized tryptophan was 10.0 μmol / ml.

  The amount of A immobilized dextran sulfate was measured by utilizing the affinity of dextran sulfate and toluidine blue. That is, about 100 ml of an aqueous solution of toluidine blue (Basic Blue 17 (Tokyo Kasei)) adjusted to about 90 mg / l is added to 3 ml of A. After stirring for 10 minutes and allowing to stand, the toluidine blue in the supernatant is measured by absorbance at 630 nm. Quantified and determined from the amount of decrease. As shown in Table 1, the amount of A immobilized dextran sulfate was 0.20 μmol / ml, and the TR / PA ratio was 50.0.

(Example 2)
To 127 ml of the same cellulose beads as in Example 1, 68 ml of water, 19 ml of 4N NaOH aqueous solution and 17.2 ml of epichlorohydrin were added and reacted by stirring at 40 ° C. for 2 hours. After the reaction, the beads were sufficiently washed with water to obtain epoxidized cellulose beads. The epoxy group amount of the epoxidized cellulose beads was 8.8 μmol / ml (wet volume).

  A dextran sulfate aqueous solution in which 7.2 g of the same dextran sulfate as in Example 1 was dissolved in 20 ml of water was prepared, 48 ml of epoxidized cellulose beads wet in water was added, and the mixture was made alkaline with an aqueous NaOH solution. Reacted for hours. After the reaction, the beads were sufficiently washed with water and brine, and then a solution prepared by dissolving 0.76 g of L-tryptophan in 25 ml of dilute aqueous NaOH was added and reacted at 50 ° C. for 8 hours. Thereafter, the beads were sufficiently washed with water and saline to obtain dextran sulfate and tryptophan-immobilized cellulose beads (B). As shown in Table 1, the tryptophan immobilized amount of B was 5.1 μmol / ml, the dextran sulfate immobilized amount was 0.28 μmol / ml, and the TR / PA ratio was 18.2.

  This B was put into a 0.2 ml test tube in the same manner as in Example 1, 3 ml of healthy human blood anticoagulated with 5 units of heparin added to 1 ml of blood, and shaken at 37 ° C. for 10 minutes. As a result of measuring the bradykinin concentration of the supernatant by radioimmunoassay, it was 2220 pg / ml.

  Moreover, B was packed in the column like Example 1, and 40 ml of healthy human blood was circulated for 2 hours. The number of blood cells in the pooled blood before and after circulation was as shown in Table 2, and all blood cells showed good passage.

(Example 3)
To 125 ml of the same cellulose beads as in Example 1, 5 ml of water, 40 ml of 4N NaOH aqueous solution and 36.3 ml of epichlorohydrin were added and reacted by stirring at 40 ° C. for 2 hours. After the reaction, the beads were sufficiently washed with water to obtain epoxidized cellulose beads. The amount of epoxy groups of the epoxidized cellulose beads was 19.9 μmol / ml (wet volume).

  A dextran sulfate aqueous solution in which 23.8 g of the same dextran sulfate as in Example 1 was dissolved in 26 ml of water was prepared, 60 ml of epoxidized cellulose beads moistened in water was added, and the mixture was made alkaline with an aqueous NaOH solution. Reacted for hours. After the reaction, the beads were sufficiently washed with water and brine, and then a solution in which 0.74 g of L-tryptophan was dissolved in 24.5 ml of dilute aqueous NaOH was added and reacted at 50 ° C. for 8 hours. Thereafter, the beads were sufficiently washed with water and saline to obtain dextran sulfate and tryptophan-immobilized cellulose beads (C). As shown in Table 1, the amount of tryptophan immobilized on C was 8.4 μmol / ml, the amount of dextran sulfate immobilized was 0.34 μmol / ml, and the TR / PA ratio was 24.7.

  This C was put into a 0.2 ml test tube in the same manner as in Example 1, 3 ml of healthy human blood anticoagulated with 5 units of heparin added to 1 ml of blood, and shaken at 37 ° C. for 10 minutes. The bradykinin concentration in the supernatant was measured by radioimmunoassay and found to be 2540 pg / ml.

  Further, C was packed in the column in the same manner as in Example 1, and 40 ml of healthy human blood was circulated for 2 hours. The number of blood cells in the pooled blood before and after circulation was as shown in Table 2, and all blood cells showed good passage.

(Comparative Example 1)
In Example 1, physiological saline was placed in a 0.2 ml test tube instead of A, 3 ml of healthy human blood anticoagulated with 5 units of heparin added to 1 ml of blood, and shaken at 37 ° C. for 10 minutes. The bradykinin concentration in the supernatant was measured by radioimmunoassay and found to be 22 pg / ml.

(Comparative Example 2)
Dextran sulfate and tryptophan-immobilized cellulose beads (D) were prepared in the same manner as in Example 2 except that the reaction time of dextran sulfate was changed from 6 hours to 0.5 hours and the amount of dextran sulfate was changed from 19.8 g to 7.2 g. ) As shown in Table 1, the tryptophan immobilized amount of D was 7.3 μmol / ml, the dextran sulfate immobilized amount was 0.07 μmol / ml, and the TR / PA ratio was 104.3.

  This D was placed in a 0.2 ml test tube in the same manner as in Example 1 and 3 ml of healthy human blood anticoagulated by adding 5 units of heparin to 1 ml of blood was added and shaken at 37 ° C. for 10 minutes. As a result of measuring the bradykinin concentration of the supernatant by radioimmunoassay, it was 25 pg / ml.

  Further, D was packed in the column in the same manner as in Example 1, and 40 ml of healthy human blood was circulated for 2 hours. The number of blood cells in the pooled blood before and after circulation is as shown in Table 2. Red blood cells showed good permeability, but leukocytes and platelets decreased to 66% and 63% before and after circulation, respectively. Somewhat bad.

(Comparative Example 3)
To 520 ml of the same cellulose beads as in Example 1, 114 ml of water, 161 ml of 4N NaOH aqueous solution and 166.4 ml of epichlorohydrin were added and stirred at 40 ° C. for 2 hours for reaction. After the reaction, the beads were sufficiently washed with water to obtain epoxidized cellulose beads. The epoxy group amount of the epoxidized cellulose beads was 16.4 μmol / ml (wet volume).

  A dextran sulfate aqueous solution in which 36.8 g of the same dextran sulfate as in Example 1 was dissolved in 104 ml of water was prepared, 245 ml of epoxidized cellulose beads moistened in water was added, and the mixture was made alkaline with an aqueous NaOH solution. The reaction was allowed for 5 hours. After the reaction, the beads were thoroughly washed with water and brine, then a solution prepared by heating and dissolving 0.80 g of L-tryptophan in 44 ml of water was added, the pH was made alkaline with an aqueous NaOH solution, and the reaction was performed at 50 ° C. for 8 hours. I let you. Thereafter, the beads were sufficiently washed with water and saline to obtain dextran sulfate and tryptophan-immobilized cellulose beads (E). As shown in Table 1, the amount of F immobilized on tryptophan was 10.0 μmol / ml, the amount of dextran sulfate immobilized was 0.14 μmol / ml, and the TR / PA ratio was 71.4.

  This E was put in a 0.2 ml test tube in the same manner as in Example 1, 3 ml of healthy human blood anticoagulated with 5 units of heparin added to 1 ml of blood, and shaken at 37 ° C. for 10 minutes. The bradykinin concentration in the supernatant was measured by radioimmunoassay and found to be 32 pg / ml.

  Further, E was packed in the column in the same manner as in Example 1, and 40 ml of healthy human blood was circulated for 2 hours. The blood counts of the pooled blood before and after circulation were as shown in Table 2, and red blood cells and platelets showed good passage.

  As is clear from Tables 1 and 2, the amount of polyanionic compound immobilized is from 0.10 μmol to 1.5 μmol per ml, and the molar ratio between the amount of tryptophan immobilized and the amount of polyanionic compound immobilized is 1. In Examples 1 to 3, which were 70 or less, the bradykinin concentration increased. However, in Comparative Example 2 and Comparative Example 3 outside these ranges, the occurrence of bradykinin was not observed as in Comparative Example 1 in which no treatment material was added.

Claims (8)

  A body fluid treatment material obtained by immobilizing a tryptophan derivative and a polyanionic compound on a water-insoluble porous carrier, wherein the immobilized amount of the polyanionic compound is 0.10 μmol or more and 1.5 μmol or less per 1 ml of the bodily fluid treatment material, A body fluid treatment material that generates bradykinin capable of whole blood treatment, wherein the molar ratio of the amount of immobilized tryptophan derivative and the amount of immobilized polyanionic compound is 1 or more and 70 or less.   The body fluid treatment material for generating bradykinin capable of whole blood treatment according to claim 1, wherein the polyanionic compound is dextran sulfate.   The body fluid treatment material for generating bradykinin capable of whole blood treatment according to claim 1 or 2, wherein the tryptophan derivative is tryptophan.   The body fluid treatment material for generating bradykinin capable of whole blood treatment according to any one of claims 1 to 3, wherein the water-insoluble porous carrier is a cellulose carrier. The body fluid treatment material for generating bradykinin capable of whole blood treatment according to any one of claims 1 to 4, wherein the exclusion molecular weight of the globular protein of the water-insoluble porous carrier is 5 x 10 ⁵ or more and 1 x 10 ⁸ or less.   A method for generating bradykinin in a body fluid, comprising contacting the body fluid treatment material for generating bradykinin capable of whole blood processing according to any one of claims 1 to 5 with the body fluid.   A body fluid for generating bradykinin capable of whole blood treatment according to any one of claims 1 to 5 in a container having an inlet and an outlet for the liquid and provided with means for preventing the body fluid treatment material from flowing out of the container. A column that generates bradykinin to the extent that whole blood processing is possible, characterized by being packed with a processing material. The column for generating bradykinin to the extent that whole blood treatment is possible according to claim 7, wherein the volume of the column is from 100 ml to 400 ml.

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