JP4893099B2 - Artificial kidney - Google Patents

Description

  The present invention relates to a hollow fiber membrane type artificial kidney that uses a high-performance hollow fiber membrane, removes lipid peroxides such as oxidized low density lipoprotein in blood, and has low endotoxin flow into the blood.

Conventionally, hollow fiber membrane blood treatment devices using hollow fiber membranes are widely used in the field of blood extracorporeal circulation, particularly in hemodialysis and plasma separation, and in particular in fields such as dialysis membranes and blood component separation membranes. Molecular hollow fiber membranes are widely used. In recent years, high-performance hollow fiber membranes with a large pore diameter that can remove a large amount of medium and high molecular weight pathogenic proteins such as β ₂ -microglobulin have become the mainstream. With the widespread use of such high-performance type hollow fiber membranes, there is a concern that endotoxin will flow into the blood from the dialysate. When endotoxin enters the blood, it causes symptoms such as fever, leukopenia, decreased blood pressure, and shock. In addition, it has been pointed out that even a trace amount below the detection limit may contribute to dialysis complications by continuously flowing into the blood.

  In addition, among long-term hemodialysis patients, findings such as decreased blood antioxidant activity and high levels of lipid peroxide have been confirmed. There is an increase in arteriosclerotic diseases. Actually, lipid peroxide cannot be removed by dialysis membranes represented by cellulose membranes, polymethyl methacrylate membranes, ethylene-vinyl alcohol membranes, polysulfone / polyvinylpyrrolidone blend membranes and the like that are commercially available. Rather, the value of lipid peroxide in blood tends to increase due to stimulation by the dialysis membrane after dialysis (Non-patent Document 1).

  Foam cells that play an important role in the formation of atherosclerotic lesions are those in which macrophages are foamed as a result of oxidatively altered lipids, especially low-density lipoproteins (hereinafter referred to as LDL), taken into macrophages. It is. Therefore, it is desired to remove lipid peroxide, particularly oxidized LDL, from the blood.

  In order to solve such problems, attention is paid to the fact that oxidized LDL has a negative charge, and methods for adsorption removal by introducing a cationic substance into the hollow fiber membrane are disclosed in Patent Document 1 and Patent Document 2. It is disclosed. As an efficient method for introducing a cationic substance into the membrane, Patent Document 3 discloses that the cationic substance is irradiated with radiation while attached to the hollow fiber membrane.

However, these methods are only intended to remove lipid peroxides such as oxidized LDL in the blood, and no consideration has been given to endotoxin present in the dialysate. Since endotoxins have a negative charge, endotoxin adsorption occurs in a membrane in which the entire membrane is excessively positively charged, resulting in a high endotoxin concentration in the vicinity of the membrane. Therefore, there is a high possibility of flowing into blood. In particular, there is a possibility that a high-performance film having high β ₂ -microglobulin removal performance may become a serious problem when it is excessively positively charged.

That is, there has never been a hollow fiber membrane type artificial kidney that removes lipid peroxides such as oxidized LDL in the blood with a high-performance hollow fiber membrane and has a low influx of endotoxin into the blood.
JP 2002-028461 A JP 2003-250885 A JP 2002-102692 A Kidney and dialysis separate volume, 38: 125 (1995)

    An object of the present invention is to provide a hollow fiber membrane type artificial kidney that uses a high-performance hollow fiber membrane, removes lipid peroxide such as oxidized LDL in blood, and has low endotoxin flow into the blood. is there.

In order to achieve the above object, the present invention has the following configuration.
(1) The zeta potential of the inner surface of the hollow fiber membrane is 20 mV to 100 mV, the total charge amount of the entire membrane is 30 μeq / g or less, and the β2-microglobulin clearance is 30 ml / min or more. Hollow fiber membrane artificial kidney.
(2) The hollow fiber membrane artificial kidney according to (1), wherein the ET permeability is 0.006% or less.
(3) The hollow fiber membrane artificial kidney according to (1) or (2), wherein the oxidized low density lipoprotein removal rate is 30% or more. (4) A cationic substance is present on the inner surface of the hollow fiber membrane. The hollow fiber membrane artificial kidney according to any one of (1) to (3), which is contained.
(5) The hollow fiber membrane artificial kidney according to any one of (1) to (4), wherein an anionic substance is contained on the outer surface of the hollow fiber membrane.
(6) The cationic substance is a primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, The hollow fiber membrane artificial kidney according to (4) or (5), which contains any one or more of a piperidine group, a pyrrolidine group, a thiazole group, and a purine group.
(7) The anionic substance contains any one or more of a carboxyl group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, and a thiocarboxyl group (5) or (6) ) Hollow fiber membrane type artificial kidney.
(8) The hollow fiber membrane artificial kidney according to any one of (1) to (7), wherein the cationic substance and the anionic substance are polymers.
(9) The hollow fiber membrane artificial kidney according to any one of (1) to (8), wherein the hollow fiber membrane has an asymmetric membrane structure having a dense layer on the inner surface.
(10) The hollow fiber membrane artificial kidney according to any one of (1) to (9), wherein the hollow fiber membrane incorporated in the artificial kidney contains a polysulfone-based polymer.

  According to the present invention, it is possible to provide a hollow fiber membrane type artificial kidney that uses a high-performance hollow fiber membrane, removes lipid peroxide such as oxidized LDL in blood, and has little influx of endotoxin into blood.

The hollow fiber membrane has a zeta potential of 15 mV to 100 mV on the inner surface of the hollow fiber membrane, a total charge amount of the whole membrane of 30 μeq / g or less, and a β ₂ -microglobulin clearance of 30 ml / min or more. It is a thread membrane artificial kidney.

  The hollow fiber membrane type artificial kidney referred to in the present invention is a module incorporating a hollow fiber membrane, and is used for hemodialysis and the like. Hemodialysis is treatment for patients with renal disease. Blood is drawn from the patient and returned to the patient's body again through the module. At that time, the module removes waste water and harmful substances and removes water by adsorption, filtration, and diffusion. In the blood of patients undergoing hemodialysis for a long period of time, it has been confirmed that lipid peroxide such as oxidized LDL has a high value, but once generated, oxidized LDL cannot be removed by dialysis. Therefore, it is removed by adsorption to the film. At this time, lipid peroxide such as oxidized LDL has a negative charge, and thus it is necessary to make the charge of the hollow fiber membrane positive. However, when the whole hollow fiber membrane is positive, there arises a problem of influx of endotoxin into the blood side.

  Since endotoxin has a negative charge, it adsorbs to a positively charged membrane. For this reason, it has been pointed out that in a positively charged membrane, endotoxin concentration in the vicinity of the membrane increases due to endotoxin adsorption, and the amount of permeation to the blood side increases.

  That is, the hollow fiber membrane is preferably a positively charged membrane in terms of adsorption and removal of oxidized LDL, and a negatively charged membrane is preferable in terms of suppression of endotoxin permeation. Here, attention was paid to the fact that the adsorption of oxidized LDL is a phenomenon occurring on the inner surface of the hollow fiber membrane, and the adsorption of endotoxin is a phenomenon occurring on the outer surface of the hollow fiber membrane. Therefore, in the present invention, a hollow fiber membrane having a positive charge on the inner surface of the hollow fiber membrane in contact with blood and a low positive charge on the entire membrane was used in order to prevent endotoxin adsorption on the membrane. As a result, it was possible to obtain a hollow fiber membrane type artificial kidney that achieves both adsorption removal of oxidized LDL and suppression of permeation of endotoxin even in a high performance hollow fiber membrane.

  The charge on the inner surface of the hollow fiber membrane can be known by measuring the zeta potential. Using a zeta potential measurement device, the pressure difference and the potential difference at both ends of the cell when the measurement liquid is made to flow through the cell are measured, and the zeta potential is obtained from the calculation based on the value and the specific conductivity of the measurement liquid. This zeta potential is a measurement result for the surface of the cell to be measured that contacts the measurement liquid. Here, the zeta potential on the inner surface of the hollow fiber membrane can be measured by using a cell in which the measurement liquid is in contact with the inner surface of the hollow fiber membrane. Details of the measurement method will be described later. In order to significantly adsorb oxidized LDL, the zeta potential on the inner surface needs to be 15 mV or more, and preferably 20 mV or more. Further, if the positive charge is too strong, platelet activation is caused and blood compatibility is deteriorated. Therefore, the zeta potential on the inner surface needs to be 100 mV or less, preferably 50 mV or less. From the above, the zeta potential on the inner surface of the hollow fiber membrane is 15 mV or more and 100 mV or less, preferably 20 mV or more and 50 mV or less.

  The total charge amount of the entire membrane is a value determined by acid / base titration method. The measuring method can be performed by a method whose details will be described later in Examples or a method according thereto. If the entire membrane is overly positive, endotoxin adsorption tends to occur. Therefore, the total charge amount is required to be 30 μeq / g or less, preferably 0 μeq / g or less. When the entire membrane is strongly negative, a negative electrolyte such as phosphorus in the blood is electrostatically repelled by the membrane, and thus the removal rate from the blood tends to decrease. Therefore, although not particularly limited, it is preferable that the total charge amount is −50 μeq / g or more.

  The method for measuring the oxidized LDL removal rate can be carried out by the method described later in detail in the examples or by a method similar thereto, but LDL is artificially oxidized and added to the blood of a healthy person, and this is added to the mini-module. From the concentration before and after recirculation at the time of recirculation, the equation (1) can be used.

r = 100 × (Ci-Co) / Ci (1)
In the formula (1), r = oxidized LDL removal rate (%), Co = oxidized LDL concentration after reflux (μg / ml), and Ci = oxidized LDL concentration before reflux (μg / ml).
A higher removal rate of oxidized LDL is preferable. A single dialysis treatment is said to increase the concentration of oxidized LDL in blood by several tens of percent or more. Therefore, it is considered that it is preferable to have a removal rate of 30% or more and further a removal rate of 40% or more in the in vitro test of the minimodule.

  The endotoxin permeability can be measured by the method described in detail later in the examples or by a method similar thereto, but the endotoxin water having a known concentration is filtered from the dialysate side of the artificial kidney to the blood side. From the concentration of endotoxin contained in the liquid that flows out from the blood side, it can be obtained by equation (2).

T = Eo ÷ Ei × 100 (2)
In the equation (2), T = endotoxin permeability (%), Eo = endotoxin concentration (EU / ml) of the liquid flowing out from the blood side, and Ei = endotoxin concentration (EU / ml) of endotoxin water.

  When endotoxin is mixed in blood, an acute reaction such as fever is caused at a high concentration, and various complications such as arteriosclerosis and anemia are caused by chronic exposure even at a low concentration. Therefore, it is preferable that the endotoxin flow into the blood side is small. Specifically, the endotoxin permeability is preferably 0.006% or less, and more preferably 0.002% or less.

  In order to make the inner surface of the hollow fiber membrane positively charged and negatively charge the entire hollow fiber membrane, it can be achieved by appropriately presenting a cationic substance on the inner surface of the hollow fiber membrane having negative charge. It can also be achieved by appropriately allowing an anionic substance to be present on the outer surface of the hollow fiber membrane having a positive charge. Alternatively, a cationic substance may be present on the inner surface and an anionic substance may be present on the outer surface.

Here, the amount of the cationic substance and the anionic substance on the surface of the hollow fiber membrane affects the charge on the inner surface of the hollow fiber membrane and the outer surface of the hollow fiber membrane. Therefore, if the amount of the substance is excessive or insufficient, as described above, problems such as a decrease in the ability to remove oxidized LDL, activation of platelets, an increase in the amount of endotoxin permeation, and a decrease in the removal amount of negatively charged substances such as phosphorus. Is concerned. Therefore, it is preferable that the cationic substance and the anionic substance are present in appropriate amounts. Although not particularly limited, in order to present an amount appropriate cationic substance and anionic substance, for example, when these materials have only one functional group, the hollow fiber membrane surface area 1 m ² per 0.0003 It is preferable to introduce 1.5 mol, and more preferably 0.00075 to 0.015 mol per 1 m ^{2 of the} surface area of the hollow fiber membrane. When the cationic substance and the anionic substance have a plurality of functional groups, the amount introduced may be adjusted depending on the type and number of functional groups. That is, when two similar functional groups are present, a half amount is a suitable value as compared with the case where there is one functional group. In addition, since the introduction efficiency and the charge differ depending on the kind of the cationic substance and the anionic substance, the introduction amount into an appropriate module may vary greatly. Also, depending on the type of hollow fiber membrane to be introduced, the proper amount of introduction may differ because the original charge of the membrane is different.

  Making the hollow fiber membrane positively charged can be achieved by the appropriate presence of a cationic substance. The presence of a cationic substance means that the cationic substance is chemically bonded or adsorbed on the hollow fiber membrane. The cationic substance here refers to a substance having an electric charge of 1 meq / g or more at pH 4.5. Among them, primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine group, pyrrolidine group, A substance having a functional group such as a thiazole group or a purine group is preferably used. From the viewpoint of availability, polyethyleneimine, polyvinylamine, polyallylamine, diethylaminoethyldextran, polylysine, polydiallyldimethylammonium chloride, a copolymer of vinylimidazolium methochloride and vinylpyrrolidone, and the like are preferably used. Two or more kinds of cationic substances may be used in combination.

  The presence of an anionic substance means that the anionic substance is chemically bonded or adsorbed on the hollow fiber membrane. The anionic substance here refers to a substance having a charge of 1 meq / g or more at pH 9.5. Among these, substances having a functional group such as a carboxyl group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, a thiocarboxyl group, and a sulfuric acid group are preferably used. From the viewpoint of availability, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, dextran sulfate and the like are preferably used. Two or more types of anionic substances may be used in combination.

  In order to obtain a hollow fiber membrane having a positive charge on the inner surface of the hollow fiber membrane and a low positive charge in the entire membrane, it can be achieved by making a large amount of the above cationic substances present on the inner surface of the hollow fiber membrane. . It can also be achieved by allowing a large amount of anionic substances to be present on the outer surface of the hollow fiber membrane. Although not particularly limited, it is preferable that the density of the cationic substance on the inner surface layer is twice or more that of the outer surface layer. Or it is preferable that the density of the anionic substance of an inner surface layer is 1/2 or less compared with an outer surface layer. The inner surface layer here refers to a region from the inner surface of the hollow fiber membrane to a depth of 3 μm, and the outer surface layer refers to a region from the outer surface of the hollow fiber membrane to a depth of 3 μm.

  The density of the substance introduced into the inner surface layer and the outer surface layer is obtained from, for example, the relative amount of the substance introduced by measuring the cross section of the hollow fiber membrane with TOF-SIMS.

  In order to make a large amount of a cationic substance or an anionic substance exist on the membrane surface, there are a method of introducing at the time of forming the hollow fiber membrane and a method of introducing after forming the hollow fiber membrane.

The hollow fiber membrane can be molded by flowing the injection solution inside when discharging the hollow fiber membrane stock solution from the double annular die, running the dry part, and then guiding it to the coagulation bath. At this time, the cationic substance can be imparted to the inner surface of the hollow fiber membrane by adding the cationic substance to the injection solution. Moreover, an anionic substance can be provided to the outer surface of the hollow fiber membrane by adding an anionic substance to the coagulation bath.

Furthermore, after incorporating the hollow fiber membrane into the artificial kidney module, a cationic substance or an anionic substance can be applied. That is, a cationic substance solution may be passed through the hollow fiber membrane inner surface side, or an anionic substance solution may be passed through the hollow fiber membrane outer surface side. Then, adsorption or chemical bonding occurs between the cationic substance or anionic substance and the surface of the hollow fiber membrane by intermolecular forces such as electrostatic interaction, hydrophobic interaction, and hydrophilic interaction.

  When the cationic substance solution is passed, it is preferable to apply filtration from the blood side to the dialysate side because it can be efficiently introduced into the inner surface of the hollow fiber membrane. In addition, it is preferable to apply filtration from the dialysate side to the blood side when passing through the anionic substance solution because it can be efficiently introduced into the outer surface of the membrane hollow fiber. Filtration here means that a substance is filtered through a membrane. The filtered state refers to a state where 1% or more, preferably 10% or more, more preferably 50% or more of the flow rate of the solution to be filtered comes out as filtrate. In the case of a hollow fiber membrane, it may be calculated from the respective flow rates on the blood side and dialysate side. At this time, as the cationic substance and the anionic substance to be used, a substance having a small molecular weight that most of the substance penetrates the membrane is unsuitable, and is preferably a polymer. That is, if an attempt is made to increase the positive charge on the hollow fiber inner surface, a large amount of a cationic substance is also introduced into the inner part of the hollow fiber membrane, so that the entire membrane is excessively positively charged. Similarly, when trying to increase the negative charge on the outer surface of the hollow fiber membrane, a lot of anionic substances are also introduced into the inner surface side of the hollow fiber membrane, so that the positive charge on the inner surface of the hollow fiber membrane is weakened. . For this reason, as the cationic substance and the anionic substance, a polymer having a weight average molecular weight of 50,000 or more, preferably 500,000 or more is suitably used.

  In addition, if a cationic substance solution or an anionic substance solution is passed through and then filtered in the same direction with a liquid that does not contain a cationic substance and an anionic substance, the cationic substance or anionic substance is efficiently introduced. Therefore, it is preferable. This is because the cationic substance or anionic substance remaining in the filling liquid is introduced to the surface of the hollow fiber membrane.

  The solvent for the cationic substance solution and the anionic substance solution is not particularly limited as long as the hollow fiber membrane does not deform, but water or alcohol is preferably used from the viewpoint of safety. The concentration of the cationic substance and the anionic substance is generally in the range of 1 wt ppm to 50 wt%, preferably 10 wt ppm to 10 wt%, depending on the type of the substance. If it exceeds 50 wt%, an introduction amount to the film corresponding to the cost cannot be obtained. On the other hand, when the amount is less than 10 ppm by weight, the amount of the cationic substance is small and the introduction efficiency is deteriorated.

  In addition, medical devices such as artificial kidneys need to be sterilized, and in recent years, radiation sterilization methods are frequently used from the viewpoint of low residual toxicity and simplicity, and γ rays and electron beams are particularly preferably used. It has been. For example, in order to sterilize a blood purification module with γ rays, it is considered that irradiation with a dose of 15 kGy or more is preferable. Moreover, radicals are generated by irradiation, and chemical bonds may be formed between the introduced substance and the hollow fiber membrane. Therefore, in such a case, the amount of the introduced substance is reduced, which is a preferable method.

  In addition, when there is a polymer that is easily denatured by γ-ray sterilization, the filling solution contains sodium pyrosulfite, ascorbic acid, ethanol, methanol, 1-propanol, isopropanol, 1-butanol, 2-butanol, glycerin, glucose, ethylene It is preferable to add a radical scavenger such as glycol.

The hollow fiber membrane is preferably a material used for medical purposes, and examples thereof include cellulose polymers, polymethyl methacrylate, polyacrylonitrile, polysulfone polymers such as polysulfone and polyethersulfone, and the like. Among these, by using a polysulfone-based polymer, a hollow fiber membrane having a high β ₂ -microglobulin clearance can be obtained, and therefore, it is preferably used.

β ₂ -microglobulin is a protein having a molecular weight of 11,500, and its accumulation causes long-term complications such as dialysis amyloidosis. At present, most commercially available artificial kidneys are type II dialysers with a β ₂ -microglobulin clearance of 10 ml / min or more. However, in recent years, high-performance artificial kidneys with a β ₂ -microglobulin clearance of 30 ml / min or more have become mainstream due to the widespread use of polysulfone-based hollow fiber membranes.

If the structure of the hollow fiber membrane is an asymmetric membrane structure having a dense layer on the inner surface, the β ₂ -microglobulin clearance can be increased, and therefore it is particularly preferably used in the present invention. Here, the asymmetric membrane structure having a dense layer on the inner surface means that the porosity in the region within 200 nm from the inner surface in the film thickness direction is 60% or less, and the porosity in the region within 200 nm in the film thickness direction from the outer surface. This refers to a hollow fiber membrane having a structure of 70% or more. As a method for calculating the porosity, an ultrathin section of a hollow fiber membrane cross section having a length of 100 nm or less in the longitudinal direction of the hollow fiber membrane is created, and an image obtained by enlarging the section by 50,000 times with a transmission electron microscope is taken. . In order to raise the contrast of an image, you may dye | stain suitably. Further, since the image obtained by the transmission electron microscope is a projected image of the sample, the void ratio is calculated to be lower as the slice to be observed becomes thicker. Therefore, it is preferable to make the slice thickness as thin as possible. The porosity in the vicinity of the surface is calculated from the obtained electron microscope image. The area for calculating the porosity is a rectangular area having a surface of 0 point and a thickness of 0 to 200 nm in the thickness direction and a width of 3 μm in the direction parallel to the surface.

The void ratio can be obtained by the expression (3) from the total area of the void portions obtained by the image processing apparatus.
Porosity = (S1 / S2) × 100 (%) (3)
In the equation (3), S1 = total area of the gap portion, and S2 = total area of the portion where image processing is performed.

  If the surface is curved or has irregularities, the most concave portion is set to 0 nm. When the surface is unclear, the surface is defined as an intermediate value between a portion having the highest contrast in the film and a portion having the lowest contrast in a non-film portion.

  The polysulfone polymer used in the present invention has an aromatic ring, a sulfonyl group and an ether group in the main chain. For example, polysulfone represented by the following chemical formulas (1) and (2) is preferably used. However, the present invention is not limited to these. N in the formula is an integer such as 50 to 80.

  Specific examples of polysulfone include Udel (registered trademark) Polysulfone P-1700, P-3500 (manufactured by Solvay), Ultrason (registered trademark) S3010, S6010 (manufactured by BASF), Victrex (registered trademark) (Sumitomo Chemical) ), Radel (registered trademark) A (manufactured by Solvay), Ultrasulone (registered trademark) E (manufactured by BASF), and the like. In addition, the polysulfone used in the present invention is preferably a polymer composed only of the repeating unit represented by the above formula (1) and / or (2). However, it does not interfere with the effects of the present invention. It may be polymerized. Although it does not specifically limit, it is preferable that another copolymerization monomer is 10 weight% or less.

    An example of a method for producing an artificial kidney will be shown. As a method for producing a hollow fiber membrane incorporated in an artificial kidney, there are the following methods. That is, a solution obtained by dissolving polysulfone and polyvinylpyrrolidone in a good solvent or a mixed solvent containing a good solvent is used as a stock solution. The polymer concentration is preferably 10 to 30 wt%, more preferably 15 to 25 wt%. The weight ratio of polysulfone and polyvinylpyrrolidone is preferably 20: 1 to 1: 5, and more preferably 5: 1 to 1: 1. As the good solvent, N, N-dimethylacetamide, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane and the like are preferable. The undiluted solution is discharged from the outer pipe of the double annular die, and after running through the dry section, it is led to the coagulation bath. An injection solution or a gas for forming a hollow portion is discharged from a tube inside the double annular base. At this time, the humidity of the dry part has an effect, so that the phase separation behavior near the outer surface is accelerated by replenishing moisture from the outer surface of the membrane while the dry part is running. It is also possible to reduce the diffusion resistance. However, when the relative humidity is too high, the solid solution coagulation on the outer surface becomes dominant, and the pore diameter is rather reduced, and as a result, the permeation / diffusion resistance during dialysis tends to increase. Therefore, the relative humidity is preferably 60 to 90%. Moreover, as an injection liquid composition, it is preferable to use what consists of a composition based on the solvent used for the undiluted | stock solution from process aptitude. For example, when dimethylacetamide is used as an injection solution concentration, an aqueous solution of 45 to 80% by weight, more preferably 60 to 75% by weight, is preferably used.

  The method of incorporating the hollow fiber membrane in the module is not particularly limited, but an example is as follows. First, the hollow fiber membrane is cut to a required length, bundled in a necessary number, and then put into a cylindrical case. Then, a temporary cap is put on both ends, and a potting agent is put on both ends of the hollow fiber membrane. At this time, the method of adding the potting agent while rotating the module with a centrifuge is a preferable method because the potting agent is uniformly filled. After the potting agent is solidified, both ends are cut so that both ends of the hollow fiber membrane are open, and a header is attached to obtain a hollow fiber membrane module.

  An example of the basic structure of the hollow fiber membrane artificial kidney obtained as described above is shown in FIG. In FIG. 1, 1 is an artery side header, 2 is a vein side header, 3 is a blood side inlet, 4 is a blood side outlet, 5 is a hollow fiber membrane, 6 is blood, 7 is a module case, 8 is a dialysate side inlet, 9 is a dialysate side outlet, 10 is a potting part, and 11 is a blood circuit.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
1. Preparation Method of Artificial Kidney Polysulfone (Udel Polysulfone (registered trademark) P-3500 manufactured by Solvay) 18 parts by weight and polyvinylpyrrolidone (BASF K30) 8 parts by weight N, N′-dimethylacetamide 73 parts by weight and water 1 In addition to parts by weight of the mixed solvent, it was dissolved by heating at 80 ° C. for 15 hours to obtain a film-forming stock solution. This film-forming stock solution was discharged from an outer tube of an orifice type double cylindrical die having an inner diameter of 0.3 mm and an inner diameter of 0.2 mm. A solution consisting of 60 parts by weight of N, N′-dimethylacetamide and 40 parts by weight of water was discharged from the inner tube as the core liquid. The discharged membrane forming stock solution passed through a dry length of 350 mm, and was then introduced into a 100% water coagulation bath to obtain a hollow fiber membrane.

10000 hollow fiber membranes obtained were inserted into a cylindrical plastic case having a dialysate inlet and a dialysate outlet as shown in FIG. 1, and both ends were sealed with resin. The sealed portion was cut so that the hollow fiber opened, and a header was attached. As described above, an artificial kidney having a hollow fiber membrane inner surface area of 1.6 m ² was prepared.
2. Measuring method 2.1. Measurement of total charge of the entire membrane The dry weight of the hollow fiber membrane was measured. At this time, 0.05 g was weighed. If the amount of charge is large and titration is not possible, the weight may be reduced as appropriate. The weighed hollow fiber membrane was washed with 20 ml of 0.1N sodium hydroxide and then with distilled water. A 1% phenolphthalein solution was dropped into the distilled water after washing, and washing with distilled water was repeated until no color was obtained. The washed hollow fiber membrane was dried until a constant weight was reached. In order to prevent heat denaturation due to drying, lyophilization is preferred, but not limited to this when freeze-drying is difficult. The hollow fiber membrane after lyophilization was placed in a 50 ml centrifuge tube. 20 ml of 0.001N hydrochloric acid was added so that the hollow fiber membrane could be immersed in hydrochloric acid. The mixture was shaken at 30 ° C. at a rate of 150 times per minute for 24 hours. 10 ml of the supernatant after shaking was titrated with 0.001N sodium hydroxide. In addition, 2 drops of 1% phenolphthalein solution was added as an indicator. The positive charge amount was determined from the titration result. Moreover, when titration was completed when the dropping amount of sodium hydroxide was less than 2 μmol, the measurement amount of the hollow fiber membrane was reduced and the measurement was performed again.

      The total charge amount was calculated from equation (4).

_{E = (V H × N H} -V N × N N) ÷ m (4)
Here, E = positively charged amount _{(μeq / g), V H} = hydrochloric acid amount (ml), normality of _{N H} = hydrochloric acid _{(μeq / ml), V N} = titre (ml), _{N N} sodium hydroxide Normality (μeq / ml), m = hollow fiber dry weight (g).
2.2. Zeta potential on the inner surface of the hollow fiber membrane 100 hollow fiber membranes were bundled and filled into a cylindrical cell having an inner diameter of 15 mm, and fixed to the end of the tube with a pot material. The pot material used here was polyurethane: KC256, KN503 manufactured by Nippon Polyurethane Industry. After fixing with the pot material, the end face was cut one day later to obtain a cell having a length of 4.5 cm to 5 cm in the height direction of the cylinder. The zeta potential was measured with a Zeta potential measuring device EKA type manufactured by Anton Peer. In this measurement, the zeta potential is calculated from the calculation by measuring the specific conductivity of the measurement liquid and the pressure difference and the potential difference at both ends of the cell when the measurement liquid is passed through the cell. The measurement liquid at that time was 0.001N potassium chloride, the measurement liquid volume was 500 ml, and the measurement pH was 2.5. Before the measurement, it was measured after being immersed in 0.001N potassium chloride overnight.
2.3. Oxidized LDL removal rate measurement 2.3.1. Production of Antioxidant LDL Antibody The one produced by Itabe et al. (H. Itabe et al., J. Biol. Chem. 269: 15274, 1994) was used. That is, human atherosclerotic lesion homogenate was injected into mice for immunization, hybridomas were prepared from the spleens of the mice, and those that reacted with copper sulfate-treated LDL were selected. The antibody class is mouse IgM and does not react with untreated LDL, acetyl LDL, or malondialdehyde LDL. Reacts with several phosphatidylcholine peroxidation products, including aldehyde derivatives of phosphatidylcholine and hydroperoxides. What was dissolved in 10 mM borate buffer (pH 8.5) containing 150 mM sodium chloride was used (protein concentration 0.60 mg / ml).
2.3.2. Preparation of oxidized LDL After desalting commercially available LDL (manufactured by Funakoshi), diluted with a phosphate buffer solution (hereinafter referred to as PBS) to 0.2 mg / ml, 1 wt% of 0.5 mM aqueous copper sulfate solution was added. And reacted at 37 ° C. for 16 hours. An oxidized LDL preparation was prepared by adding 25 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) to a concentration of 1 wt%, 10 wt% sodium azide to 0.02 wt%.
2.3.3. Adsorption Operation The oxidized LDL was added to healthy human plasma (Japanese, 30s) to 2 μg / ml.

  A mini-module with a length of 12 cm and a number of 50 is made of hollow fiber membranes, and an inner diameter of 0.8 mm (outer diameter) through a silicone tube (product name ARAM) with an inner diameter of 7 mm (outer diameter of 10 mm) and a length of 2 cm and a deformed connector. A silicone tube (diameter 1 mm) (product name: ARAM, 37 cm, two at each end) was connected, and 1.5 ml of the plasma was circulated in the hollow fiber at 25 ° C. for 4 hours at a flow rate of 0.5 ml / min.

Furthermore, the recirculation operation was performed only with the silicone tube without attaching the mini module.
2.3.4. Measurement of oxidized LDL concentration Antioxidant LDL antibody was diluted to 5 μg / ml with PBS, dispensed into a 96-well plate at 100 μl / well, shaken at room temperature for 2 hours, and then adsorbed on the wall at 4 ° C. overnight or longer. I let you.

  Discard the antibody solution in the well, dispense 200 μl / well of Tris-hydrochloric acid buffer (pH 8.0) containing 1 wt% Bovine Serum Albumin (BSA. “Fraction V”, Seikagaku Corporation), and then at room temperature for 2 hours. After permeation and blocking the wall, discard BSA in the well, and distribute 100 μl / well of plasma containing oxidized LDL and a standard for preparing a calibration curve (PBS buffer containing 0-2 μg / ml oxidized LDL). did. Then, after permeating at room temperature for 30 minutes, it was left at 4 ° C. overnight.

  After returning to room temperature, the solution in the well was discarded, and the well was washed three times with Tris-HCl buffer (pH 8.0) containing 0.05 wt% “Tween-20” (Katayama Chemical). Sheep anti-apo B antibody (THEBINDING SITE) diluted 2000 times with PBS was dispensed at 100 μl / well into the washed wells, and permeated at room temperature for 2 hours. Then, anti-apo B antibody in the wells was discarded, and 0.05 wt. The wells were washed three times with Tris-HCl buffer (pH 8.0) containing% “Tween-20” (Katayama Chemical). 100 μl / well of alkaline phosphatase-labeled donkey anti-sheep IgG antibody (CHEMICON) diluted 2000 times with Tris-HCl buffer (pH 8.0) containing 2 wt% “Block Ace” (Dainippon Pharmaceutical) in the washed well The plate was washed twice and further washed twice with Tris-HCl buffer (pH 8.0). Subsequently, 1 mg / ml solution (0.0005M magnesium chloride, 1M diethanolamine buffer, pH 9.8) of p-nitrophenyl phosphate (Boehringer Mannheim GmbH) was dispensed at 100 μl / well and reacted at room temperature for an appropriate time. Then, the absorbance at 415 nm was measured with a plate reader. A calibration curve was drawn from the standard results to determine the oxidized LDL concentration.

The oxidized LDL removal rate was calculated from the above formula (1) by quantifying oxidized LDL in plasma before and after reflux. The oxidized LDL removal rate with only the silicone tube was calculated and subtracted.
2.4. Endotoxin permeability measurement Influx of endotoxin from the dialysate side to the blood side is indicated by endotoxin permeability. Here, the endotoxin permeability is a percentage value of endotoxin that has flowed to the dialysate side and endotoxin that has flowed to the blood side. A lower endotoxin permeability is a better artificial kidney because there is less endotoxin flow into the blood. The endotoxin permeability test method is shown below. Endotoxin-free water (Japanese Pharmacopoeia water for injection: Otsuka Pharmaceutical) 1 L (flow rate 500 ml / min) was introduced from the blood outlet 4 and out of the blood inlet 3 to wash the module. At this time, the dialysate outlet was plugged. Next, 1 L of endotoxin-free water (flow rate 500 ml / min) was introduced from the blood outlet 4 and out of the dialysate outlet 9 to wash the module. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged.

  Into the washed module, 15 EU / mL endotoxin water was introduced from the dialysate inlet 8 and exited from the blood inlet 3. At this time, the blood outlet 4 and the dialysate outlet 9 were plugged. 15 L was flowed at a flow rate of 500 ml / min, and the final 1 mL was collected to measure the endotoxin concentration. The solution was passed at room temperature, and the temperature of the endotoxin water was 25 ° C. A polystyrene test tube (Falcon) was used for the collection tube. The collected sample has a low endotoxin concentration, and therefore, adsorption and deactivation are likely to occur. For this reason, it is necessary to use polystyrene, which is a material that hardly adsorbs endotoxin, and it is necessary to measure endotoxin immediately after collection. It is desirable to perform measurement within 10 minutes after collection.

  The endotoxin water before passing the module is divided by the endotoxin concentration after passing through the module and multiplied by 100 to obtain the endotoxin permeability. The method for measuring the endotoxin concentration is shown below. 200 μL of a sample was placed in a dialysate Limulus reagent (dialysate Limulus reagent for 0.2 ml: Wako Pure Chemicals) and vortexed for 3 seconds. The gelation time of the Limulus reagent was measured with a toxinometer (ET-301: manufactured by Wako Pure Chemical Industries, Ltd.). At this time, the set temperature is 37 ° C. and the measurement time is 200 min. In the same manner, an endotoxin standard (derived from Wakken E. coli UKT-B) was measured and used as a calibration curve.

Endotoxin permeability was calculated from the above formula (2).
2.5. β ₂ -microglobulin clearance measurement method β ₂ -microglobulin was added to bovine serum adjusted to 37 mg at 5 mg / l. The solution was allowed to flow at 1 ml / min to the blood side of a glass tube module (a module having an effective length of 100 mm in which both ends were fixed with an adhesive through 36 hollow fibers in a glass tube) in a 37 ° C. water bath. At the same time, PBS (−) (Nissui Pharmaceutical Dulbecco PBS (−)) powder of 9.6 g dissolved in 1 L of distilled water was flowed from the dialysate side at a flow rate of 20 ml / min. After circulating for 2 hours, whole blood serum was collected and frozen in a freezer at -20 ° C or higher. The clearance was calculated from the formula (5) from the concentration of β ₂ -microglobulin in each solution.

Co = (CBi−CBo) × Q _B / CBi (5)
Here, C _O = β ₂ -microglobulin clearance (ml / min), CBi = module inlet side concentration (mg / l), CBo = module outlet side concentration (mg / l), Q _B = module supply liquid amount ( ml / min).
Example 1
Polyethyleneimine was used as the cationic substance. Polyethyleneimine (Sigma Mw 750,000) 100 ppm by weight aqueous solution is introduced from the blood outlet 4 of the artificial kidney obtained above, and from the dialysate outlet 9 to introduce polyethyleneimine through filtration into the hollow fiber membrane. did. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged. The flow rate was 500 mL / min and the flow rate was 1 L. Subsequently, water was introduced from the blood outlet 4 and exited from the dialysate outlet 9, and the entire amount of polyethyleneimine was introduced into the hollow fiber membrane by filtration. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged. The flow rate was 500 mL / min and the flow rate was 2 L. The filling was carried out at room temperature, and the polyethyleneimine aqueous solution was carried out at a liquid temperature of 20 ± 5 ° C. Both the polyethyleneimine aqueous solution and water are filtered from the blood side to the dialysate side. Thereafter, the module was irradiated with γ rays. The amount of γ-ray irradiation was 27 kGy. This was followed by an endotoxin permeability test. Similarly, a module is prepared, the hollow fiber membrane of the module is cut out, β ₂ -microglobulin clearance is measured, the oxidized LDL removal rate is measured, the total charge of the entire membrane is measured, and the inner surface of the hollow fiber membrane The zeta potential at pH 2.5 was measured. The result was as shown in Table 1. That is, by forming a hollow fiber membrane having a negative total charge amount and a high zeta potential at pH 2.5 on the inner surface of the hollow fiber membrane, removal of oxidized LDL even in a membrane with high β ₂ -microglobulin clearance A hollow fiber membrane having excellent rate and low endotoxin permeability could be obtained.
Comparative Example 1
Polyethyleneimine was used as the cationic substance. Polyethyleneimine (Sigma Mw 10,000) 1 wt% aqueous solution is introduced from the blood outlet 4, the blood inlet 3 is connected to the dialysate inlet 8, and the dialysate outlet 9 is exited. Introduced inside and outside. The flow rate was 200 mL / min, and the mixture was circulated for 20 minutes. The filling was carried out at room temperature, and the polyethyleneimine aqueous solution was carried out at a liquid temperature of 20 ± 5 ° C. Thereafter, the module was irradiated with γ rays. The amount of γ-ray irradiation was 27 kGy. This was followed by an endotoxin permeability test. Similarly, a module is prepared, the hollow fiber membrane of the module is cut out, β ₂ -microglobulin clearance is measured, the oxidized LDL removal rate is measured, the total charge of the entire membrane is measured, and the inner surface of the hollow fiber membrane The zeta potential at pH 2.5 was measured. The result was as shown in Table 1. That is, by making a hollow fiber membrane having a high zeta potential at pH 2.5 on the inner surface of the hollow fiber membrane, the oxidation LDL removal rate is excellent, but the total charge amount of the entire membrane is positive, so that the endotoxin permeability is high. Has become a high hollow fiber membrane.
(Comparative Example 2)
AN69ST (Polyacrylonitrile, Hospal)
The endotoxin permeability test was conducted. Further, a hollow fiber membrane of AN69ST (manufactured by Hospal) was cut out, β ₂ -microglobulin clearance measurement, oxidized LDL removal rate measurement, total charge amount measurement of the whole membrane, and pH of the inner surface of the hollow fiber membrane. The zeta potential at 5 was measured. The result was as shown in Table 1. The zeta potential on the inner surface of the hollow fiber membrane is high, and the oxidized LDL removal rate is excellent. However, since the total charge amount of the entire membrane is strong positive, it is a hollow fiber membrane having a high endotoxin permeability even though the membrane has a low β ₂ -microglobulin clearance.
(Comparative Example 3)
Water was introduced into the artificial kidney from the blood outlet 4 and out of the dialysate outlet 9. The flow rate was 500 mL / min, and the flow rate was 0.5 L. At this time, the blood inlet 3 and the dialysate inlet 8 were plugged. Filling was performed at room temperature, and water was performed at a liquid temperature of 20 ± 5 ° C. Thereafter, the module was irradiated with γ rays. The amount of γ-ray irradiation was 27 kGy. This was followed by an endotoxin permeability test. Similarly, a module is prepared, the hollow fiber membrane of the module is cut out, β ₂ -microglobulin clearance is measured, the oxidized LDL removal rate is measured, the total charge of the entire membrane is measured, and the inner surface of the hollow fiber membrane The zeta potential at pH 2.5 was measured. The result was as shown in Table 1. The zeta potential on the inner surface of the hollow fiber membrane is low, and the oxidation LDL removal rate is poor. However, since the total charge amount of the entire membrane is low, the endotoxin permeability is a hollow fiber membrane.

1 shows an embodiment of an artificial kidney used in the present invention.

Explanation of symbols

1. 1. Arterial header 2. Venous header 3. Blood side inlet 4. Blood side outlet Hollow fiber membrane6. Blood 7. Module case 8. Dialysate side inlet 9. Dialysate side outlet 10. Potting unit 11. Blood circuit

Claims (10)

A hollow fiber membrane having a zeta potential on the inner surface of the hollow fiber membrane of 20 mV to 100 mV, a total charge amount of the whole membrane of 30 μeq / g or less, and a β2-microglobulin clearance of 30 ml / min or more. Type artificial kidney.   The hollow fiber membrane artificial kidney according to claim 1, wherein the endotoxin permeability is 0.006% or less.   The hollow fiber membrane artificial kidney according to claim 1 or 2, wherein the removal rate of oxidized low density lipoprotein is 30% or more.   The hollow fiber membrane artificial kidney according to any one of claims 1 to 3, wherein a cationic substance is contained on the inner surface of the hollow fiber membrane.   The hollow fiber membrane type artificial kidney according to any one of claims 1 to 4, wherein the outer surface of the hollow fiber membrane contains an anionic substance.   The cationic substance is a primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine group, 6. The hollow fiber membrane artificial kidney according to claim 4 or 5, comprising any one or more of a pyrrolidine group, a thiazole group, and a purine group.   The hollow according to claim 5 or 6, wherein the anionic substance contains at least one of a carboxyl group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, and a thiocarboxyl group. Thread membrane artificial kidney.   The hollow fiber membrane artificial kidney according to any one of claims 1 to 7, wherein the cationic substance and / or anionic substance is a polymer.   The hollow fiber membrane artificial kidney according to any one of claims 1 to 8, wherein the hollow fiber membrane has an asymmetric membrane structure having a dense layer on the inner surface.   The hollow fiber membrane type artificial kidney according to any one of claims 1 to 9, wherein the hollow fiber membrane incorporated in the artificial kidney contains a polysulfone polymer.

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