JP3259860B2 - Removal device for albumin-bound bilirubin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for efficiently adsorbing and removing bilirubin bound to albumin in plasma.

[0002]

2. Description of the Related Art In general, liver failure, which is seen as a terminal symptom of liver disease, has a remarkably high plasma bilirubin concentration. Therefore, for the purpose of treating hepatic failure, blood purification therapy in which patient blood or plasma is passed through a container filled with activated carbon to adsorb bilirubin in patient plasma has been widely performed. However, blood purification therapy using activated carbon has a low ability to adsorb bilirubin, and how to increase the ability to adsorb bilirubin has been an important issue.

On the other hand, an adsorbent composed of a water-insoluble porous material having a basic functional group on the surface has a higher adsorption selectivity for bilirubin and an excellent adsorption capacity for bilirubin as compared with the above-mentioned activated carbon. Is widely used as an adsorbent for bilirubin in the field of adsorption therapy for dentistry (for example, JP-A-54-135497, JP-A-55-135497).
106165). However, the bilirubin adsorption capacity is not satisfactory, and it is strongly desired to further increase the adsorption capacity.

[0004] It is known that bilirubin in plasma includes a form bound to albumin and a form not bound to albumin. Attempts have been made to increase the ability of total bilirubin to be adsorbed and removed by weakening or dissociating the bound bilirubin with albumin and then adsorbing bilirubin with activated carbon. For example, benzoic acid is added to plasma to be purified in advance as a dissociating agent for bilirubin from albumin, and then the plasma is adsorbed with bilirubin using activated carbon as an adsorbent, thereby enhancing the adsorption of bilirubin to activated carbon. (Tra
ns Am Soc Artif Intern Or
gans, 34, 585-589, 1988).

[0005]

Activated carbon is excellent in adsorbing many low molecular weight compounds. For this reason, when adsorbing bilirubin with activated carbon, an attempt to add benzoic acid to blood beforehand has a problem that activated carbon adsorbs benzoic acid itself in a large amount. By adding acid to the advance in the blood as bilirubin dissociation agent from albumin, although increases the adsorption capacity of bilirubin against the active carbon, simultaneously, the added acid may, thus immediately adsorbed and removed upon contact with the activated carbon . It is thought that benzoic acid reacts with and binds to albumin to release bilirubin from albumin and promotes the adsorption of bilirubin to activated carbon.As a result, the adsorption capacity for activated carbon is thought to be increased. Is inhibited by activated carbon and does not efficiently act on the release of bilirubin from albumin. For this reason, in the above-mentioned trial, the activated carbon is pretreated in advance with benzoic acid to adsorb a large amount of benzoic acid. However, pretreatment of activated carbon with benzoic acid greatly reduces the total adsorption capacity of activated carbon, and is economically problematic and complicated.

[0006] Furthermore, activated carbon is excellent as a nonspecific adsorbent for low molecular weight substances, but is insufficient as a selective adsorbent for bilirubin for purifying blood or plasma in terms of selectivity and total adsorption capacity of bilirubin. Met. Although the adsorption capacity is increased by adding benzoic acid to plasma in advance, more excellent bilirubin adsorption capacity is required, and the adsorption selectivity is not improved at all. Adsorbents having a basic functional group on the surface are superior to activated carbon in terms of the selectivity of adsorption to bilirubin and the total adsorption capacity, but there is a strong demand for higher performance especially in terms of the adsorption capacity. An object of the present invention is to provide a safe blood circulation and treatment method which has excellent selectivity for adsorption and can adsorb and remove bilirubin in plasma much more efficiently than before.

[0007]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies to establish a method for removing bilirubin which does not have the above-mentioned problems and is more excellent in removing ability, and as a result, the present invention has been accomplished. . That is, the present invention provides a plasma reservoir (8) for supplying plasma or
Is a plasma separator (5), a plasma pump (6) and non-activated carbon
Means for removing bilirubin (1) and means for removing dissociating agent (2)
Are placed in this order from upstream to downstream.
Equipped with plasma pump (6) and non-activated carbon-based bilirubin
Dissociation agent reservoir (3)
The dissociation agent supply flow path with the release agent mixing pump (4) is connected
An apparatus for removing albumin-bound bilirubin, comprising:

[0008] The means for removing free bilirubin referred to herein includes means for separation by molecular size using a membrane or the like;
For example, in addition to filtration, dialysis, and the like, means by adsorption using an adsorbent may be mentioned, and any method can be used. The removal method based on the molecular size using a membrane or the like is useful in that the dissociating agent used at the same time as the removal of bilirubin can be removed more slowly than the adsorption with activated carbon, but it is also possible to remove useful substances having a lower molecular weight than bilirubin. There is a disadvantage. Therefore, the method using an adsorbent is more preferable because the selectivity to bilirubin is higher than the membrane method, there is no removal of useful low-molecular-weight substances, the handling is excellent, and no replacement fluid is required.

The dissociating agent can be used in a solid state such as a powder or a particle. However, when it is mixed with blood or plasma, it is preferable that the dissolving agent be in a solution state, since it can be mixed well, and an oily substance such as cottonseed oil can also be used. Is preferably an aqueous solution in that it does not affect biological components such as plasma proteins and blood cell components. The mixing amount of the solution in which the dissociating agent is dissolved with respect to the plasma (dissociation solution) is preferably not more than 1/2 volume of the plasma volume, more preferably not more than 1/5 volume, in order to avoid dilution of plasma components such as proteins. is there. To avoid dilution, the smaller the amount of dissociation solution to be added, the better.However, in blood purification therapy, it is necessary to mix the dissociation solution with the dissociation solution without stagnation of blood or plasma in the process of circulating blood or plasma. It is. If the amount of the dissociating solution with respect to blood or plasma is too small, the dissociating agent may not be sufficiently mixed. Therefore, the volume of the dissociated solution mixed is 1/1 of the plasma volume.
It is preferably at least 00 volume, more preferably 1 /
More than 50 volumes. Mixing of the dissociation solution with plasma can be performed intermittently, but it is desirable to continuously administer and mix the dissociation solution from the viewpoint of more uniform mixing.

If the mixing amount of the dissociating agent is too small, a sufficient effect cannot be obtained, and if the mixing amount is too large, a high effect can be obtained, but many dissociating agents are required, which is economically undesirable.
It is generally desirable that the amount of the dissociating agent remaining in the plasma after the adsorption treatment is also small. In our studies, it was preferred that the concentration of the dissociating agent after mixing with plasma be 1 mM or higher. More preferably, it was 4 mM or more, and still more preferably 10 mM or more. The upper limit is preferably 100 mM or less. A high effect is often obtained with a smaller amount of the dissociating agent, and therefore a dissociating agent concentration of 10 mM or more and 50 mM or less is most preferable.

Although the removal apparatus of the present invention can be applied to plasma in whole blood, considering the bilirubin-adsorbing ability of a known bilirubin adsorbent, it can be separated from whole blood in advance by centrifugation or membrane separation. It is preferable to apply to plasma which has been obtained. That is, at this time, eliminating the mixing of the dissociating agent into the plasma on the blood cell side after the plasma separation, reducing the amount of the dissociating agent, and improving the uniformity of mixing of the dissociating agent,
It is more preferable to mix a dissociating agent into the plasma after removing the blood cell components. As a method for separating plasma, a membrane separation method is preferable in terms of economic efficiency and no contamination of blood cell components. As shown in FIG. 1, whole blood sent to the plasma separator 5 by the pump 7 is separated into plasma and blood cells. Plasma separation membrane 5 and bilirubin adsorbent 1
During this period, a method of mixing the dissociating agent with the plasma by continuously injecting the dissociating agent 3 into the plasma is particularly preferable. The method of mixing the dissociation agent with the plasma is to mix the dissociation agent from the side of the plasma flow path to eliminate the stagnation and resistance of the plasma, or the residence time of the plasma in the plasma reservoir is 1 minute. In the following cases, it is also preferable that the plasma does not undergo coagulation due to stagnation, so that the plasma reservoir is provided and mixed with the dissociating agent in the plasma reservoir.

In the membrane separation method, in order to control the amount of plasma collected,
A plasma pump 6 may be used between the plasma outlet of the membrane separator 5 and the bilirubin adsorbent inlet. At this time, it is preferable that the dissociating agent mixing site 13 is provided on the adsorbent side of the plasma pump, that is, on the outlet side of the plasma pump, since the risk of excessive injection of the dissociating agent can be avoided.

It is necessary to keep the albumin-bilirubin complex and the dissociating agent in the plasma in a mixed state for a certain period of time after mixing the plasma and the dissociating agent. May be performed simultaneously in the same device. According to the study of the present inventors, after mixing the dissociating agent and the albumin bilirubin complex, a good effect is not obtained immediately in terms of adsorption of bilirubin to the bilirubin adsorbent, but a certain reaction time is required. Met. The adsorption capacity gradually increased from the start of the reaction between the mixture of plasma and the dissociating agent and the bilirubin adsorbent, and became constant in about 5 minutes.
The time from the start of the reaction between the dissociating agent and the albumin / bilirubin complex to the end of the reaction between the mixture and the bilirubin adsorbent is less than 30 seconds, a sufficient effect cannot be obtained, and the time is preferably 1 minute or more. It was good. It is particularly preferable that after the plasma and the dissociating agent are mixed, it takes 5 minutes or more until the reaction with the bilirubin adsorbent is completed. The upper limit of the time is not particularly limited, and may be any as long as blood does not stay for a long time.

The dissociating agent used in the present invention may be any one capable of reducing the binding strength between albumin and bilirubin.
Any of them can be used, but preferred examples include compounds having a negative charge in plasma and compounds known as chaotropic substances that weaken hydrophobic bonds. Among compounds having a negative charge in plasma, compounds having both a hydrophobic portion and a negative charge in one molecule, such as fatty acids and aromatic acids, are more preferable. Specific examples of these include benzoic acid, phthalic acid, phenol, styrenesulfonic acid, oleic acid, linoleic acid,
Linolenic acid, malonic acid, citric acid, ethylene glycol, propylene glycol, glycerin, sodium thiocyanate and the like. Further, aspirin, phenylbutazone, oxyphenylbutazone, ketophenylbutazone, mefenamic acid, flufenamic acid, ibufenac, ibuprofen, alclofenac,
Such as ketoprofen, indomethacin, clidanac, hydrocortisone, prednisolone, dexamethasone, sulfizoxazole, sulfamethoxazole, sulfamethizole, gentamicin, lincomycin, benzylpenicillin, bibumecilinam, cefotium, warfarin, tolbutamide, phenytoin, etc. Steroid anti-inflammatory drugs, antibiotics, sulfa drugs, anticoagulants, oral hypoglycemic drugs, antiepileptic drugs, and dyes such as bathoflavin, indocyanan green, indivamide, promocresol green, flavaspidic acid, and phenol red Can also be given. Among these, particularly preferred examples of bilirubin release include benzoic acid, mefenamic acid, flufunamic acid, ibfenac, clidanac, propylene glycol, sodium thiocyanate and the like.

The albumin-bilirubin complex was the term Al blanking Min in the plasma of the present invention include mercapto albumin and non mercaptalbumin and albumin have undergone various in vivo mutation such as glycosylase late albumin. The bilirubin includes glucuronic acid inclusion and non-inclusion. The mode of binding between albumin and bilirubin is ionic and / or hydrophobic, excluding those linked covalently.

According to the study of the present inventors, it has been found that bilirubin and the dissociating agent differ greatly in the pore diameter on the adsorbing adsorbent. While the dissociating agent is adsorbed on the entire pore size of the adsorbent, bilirubin is adsorbed competitively with the dissociating agent or the like on a larger pore size. Therefore, it is suitable for a bilirubin adsorbent to reduce the surface area of small pores that do not adsorb because bilirubin cannot enter, and increase the surface area of pores that can adsorb bilirubin. In particular, bilirubin is adsorbed on pores having a pore diameter of 100 ° or more, while dissociating agents are adsorbed particularly well on pores having a pore diameter of 100 ° or less. Activated carbon has a very large surface area of pores having a pore diameter of 200 ° or less, particularly 100 ° or less. Therefore, the dissociating agent is quickly adsorbed on activated carbon, and the effect of the dissociating agent cannot be obtained.

Adsorbents suitable as bilirubin adsorbents include:
The surface area of the pores having a pore diameter of 200 ° or less is preferably 10 m ² or less because the dissociating agent is hardly adsorbed, and the surface area of the pores having a diameter of 100 ° or less is more preferably 5 m ² / ml or less. . Further, from the viewpoint of bilirubin adsorption ability, the average pore diameter is 100 ° or more, more preferably 400 ° or more.
Å or more, and the total pore surface area is 1 m ² / ml or more, preferably 2 m ² / ml or more, more preferably 10 m ² / ml or more.
m ² / ml or more, and most preferably 20 m ² / ml or more. In particular, the pore diameter 20 relative to the total pore surface area
The ratio of the pore surface area of 0% to 2,000% is 10%.
It is preferably at least 30%, more preferably at least 30%. Most preferably, it is 50% or more.

The bilirubin adsorbent may adsorb bilirubin by either hydrophobic interaction or electrostatic interaction. The electrostatic interaction is more preferable than the hydrophobic interaction because the effect extends farther than the surface. According to the study of the present inventors, in particular, the dissociating agent such as benzoic acid added to the surface of the water-insoluble porous material surface, in that it efficiently reacts with albumin without adsorbing on the bilirubin adsorbent and liberates bilirubin. It has been found that the adsorbent having a functional group is very excellent.

Heparin is the most widely used anticoagulant when adsorbing and removing bilirubin from plasma using a bilirubin adsorbent. However, since heparin has an acidic functional group in the molecule, there is a problem that it is electrostatically adsorbed to the basic functional group of the bilirubin adsorbent. Since heparin is a mixture of various molecular weights, it is unlikely that it can be completely eliminated at a specific pore size.However, heparin is considered to have a higher anticoagulant effect with a higher molecular weight, and partially adsorbs low molecular weight heparin However, it is practically preferable to make the pore diameter so as not to adsorb high molecular weight substances.

According to the study of the present inventors, it has been found that, when the pore diameter of the pores is 1,000 ° or more, particularly more than 2,000 °, the adsorption capacity of bilirubin is high, but the heparin adsorption is also high. Therefore, the bilirubin adsorbent used in the present invention desirably has an average pore diameter of 2,000 ° or less, more preferably 1600 ° or less, from the viewpoint of reducing the above-mentioned adsorption rate of heparin. The most preferred upper limit of the average pore diameter is 1,200 °. At this time, the ratio of the surface area of the pores having a pore size of 2,000 to 80,000 to the surface area of the pores having a pore size of 100 to 2,000 is preferably 40% or less, more preferably 20%. It was below. Similarly, from the point of bilirubin adsorption capacity and non-specific adsorption of heparin, when the pore diameter at which the differential value is the maximum in the differential pore distribution based on the pore volume is defined as 100 °,
Or more, more preferably 500 ° or more. At this time, the upper limit is not more than 5,000, preferably not more than 2,000 °, more preferably not more than 1,600 °, and furthermore not more than 1,20 °.
0 ° or less.

From the viewpoint of suppressing the adsorption of the dissociating agent and heparin and obtaining a high bilirubin adsorption effect, it is often an important requirement that the surface area is concentrated in pores having a pore diameter of 100 to 2,000 mm as much as possible. When the ratio of the surface area of the pores having a pore diameter of 100 to 2,000 mm relative to the total pore surface area is shown, it is preferably 10% or more. More preferably 30%
Or more, particularly preferably 50% or more. When the distribution of the pores is shown on a volume basis, the pore diameter is 100 ° or more and 2,000.
The volume of the pores of not more than 60% is not less than 30%, more preferably not less than 40% of the total pore volume of not less than 60 ° and not more than 80,000 °,
It is more preferably 50% or more, and the volume of pores having a pore size of 2,000 to 80,000 is 60% or less.
The volume is preferably 50% or less, more preferably 40% or less of the total pore volume of {80,000} or less. At this time, the ratio of the volume of the pores having a pore size of 2,000 to 80,000 to the volume of the pores having a pore size of 100 to 2,000 is 120% or less, preferably 100% or less.

Methods for measuring the surface area of the porous body include a physical adsorption method, a heat of immersion method, a permeation method, and a chemical adsorption method. The mercury intrusion method used for measuring the distribution of the pore diameter and the pore volume of the porous body is used. Is also required. The mercury intrusion method is useful because a surface area for each pore diameter is obtained. Here, pores of 60 ° or less are difficult to measure accurately by the mercury intrusion method,
Since the pores exceeding 0 ° have extremely small surface area and have substantially negligible effect on adsorption, the pore diameter is 60 ° or more and 8% or less.
Those having a diameter of not more than 000 ° were treated as pores.

The pores referred to here are preferably values in a state close to practical use as much as possible. When the surface has a coating layer, the value after coating treatment is used. When the shape changes due to the drying process at the time of measurement by the mercury intrusion method, the change in particle size is measured, and the surface area is calculated by multiplying the rate of change in particle size by the square and the pore volume by the cube of the particle size. It was decided to correct it. That is, when the particle diameter is 1 / X times, the surface area is 1 / X ² times and the pore volume is 1 / X ³ times. Specifically, correction based on the apparent specific gravity and the swelling ratio is performed as necessary.

In the present invention, a means for removing the dissociating agent from the plasma after removing bilirubin from the mixture of the plasma and the dissociating agent is employed in order to avoid the influence of the dissociating agent on blood cell components and other biological components. More desirable. The dissociation agent can be removed by a method such as filtration or dialysis using a membrane because the dissociation agent is a low molecular weight substance. The use of an agent is preferred in that no replacement fluid is required.

The adsorbent of the dissociating agent preferably has a pore diameter of 200 ° or less and a pore surface area of 10 m ² or more from the viewpoint of the adsorbing ability of the dissociating agent. At this time, it is more preferable that the pore surface area of 100 ° or less is 5 m ² or more. In particular, the surface area of the pores having a pore diameter of 200 ° or less is preferably 20 m ² or more, and the surface area of the pores having a pore diameter of 100 ° or less is most preferably 10 m ² or more. From the viewpoint of reducing non-specific adsorption of substances other than the dissociating agent such as plasma protein and increasing the selectivity of adsorption to the dissociating agent, the ratio of pores having a pore diameter of 200 mm or less to the total pore surface area is preferably 50% or more, particularly Most preferably, it is 80% or more. Alternatively, the pore diameter 100 relative to the total pore surface area
The ratio of pores of Å or less is preferably 30% or more, particularly 50% or less.
% Or more is more preferable.

As the adsorbent for the dissociating agent, any adsorbent having the above physical structure, such as physical adsorption such as hydrophobic interaction and adsorption by electrostatic interaction, can be used.
In particular, a porous material having a hydrophobic surface or a material having a carbonized surface such as activated carbon is preferable. Among them, activated carbon is a very excellent adsorbent for low molecular weight compounds, and is useful because the dissociating agent used in a small amount of activated carbon can be efficiently adsorbed. Since the amount used here may be smaller than that in the case of using activated carbon for bilirubin adsorption, non-specific adsorption, which was a problem of activated carbon, can be reduced and can be avoided.

The means for removing the dissociating agent may be located at any position as long as it is performed after the means for removing bilirubin. However, bilirubin is removed from the plasma after the blood cell component and the plasma are separated in advance, and When mixing with the blood cell component again, it is preferable to perform the mixing before mixing with the blood cell component because the removal efficiency of the dissociation agent is high and the influence of the dissociation agent on the blood cell component can be avoided. Also, if the plasma comes into contact with the adsorbent of the dissociating agent after contacting with the adsorbent of bilirubin, the adsorbent of the bilirubin adsorbent and the adsorbent of the dissociating agent may be filled in separate containers, or in the same container. For example, they may be filled in layers.

The basic functional group on the surface of the bilirubin adsorbent includes amines, amine derivatives and the like.
Examples thereof include a quaternary amine group and a quaternary ammonium group, and any of them may be used. Preferred examples include those having a pKa of 4.0 or more. The basic functional group does not necessarily need to be present on the side chain of the polymer chain constituting the water-insoluble porous body, and may be one that forms the main chain. However, it is easier to introduce a basic functional group as a side chain into the water-insoluble porous body in terms of production.

When the amount of the basic functional group of the bilirubin adsorbent, that is, the ion exchange capacity (measured by the neutral salt decomposition method) is small, the practical ability as a bilirubin adsorbent for purifying blood or plasma is poor, and If the ion exchange capacity is too large, non-selective adsorption tends to increase slightly. Therefore, a preferable ion exchange capacity is 0.01 m
The range is from eq / ml to 100 meq / ml, more preferably from 0.1 meq / ml to 50 meq / ml.

The water-insoluble porous material constituting the bilirubin adsorbent is not limited to an inorganic compound or an organic compound, but has a small amount of eluted substances with respect to warm water, and can easily and precisely control the pores of the porous material. Organic polymers are preferred. In particular, a porous body into which a basic functional group can be introduced is preferable.
Examples of such polymers include polymers and copolymers of vinyl compounds such as polypropylene, polystyrene, polymethacrylate ester, polyacrylate ester, polyacrylic acid and polyvinyl alcohol, polyamide compounds such as nylon 6 or 66, and polyethylene terephthalate. And polyester-based compounds such as cellulose and plant-derived polysaccharide-based compounds such as cellulose.

The porous material used in the present invention into which a basic functional group can be introduced has a basic functional group of 0.01 m
It is sufficient that eq / ml or more can be introduced, and it is not limited to the above. In the examples, polymers and copolymers of vinyl compounds are more preferably used because of ease of polymerization and ease of introduction of a basic functional group. Such examples include styrene, methyl styrene, diphenyl ethylene, ethyl ethylene, dimethyl styrene, vinyl naphthalene, vinyl phenanthrene, vinyl mesitylene,
Hydrocarbon compounds such as 4,6-trimethylstyrene and 1-vinyl-2-ethylacetylene: chlorostyrene, methoxystyrene, bromostyrene, cyanostyrene, fluorostyrene, dichlorostyrene, N, N-dimethylaminostyrene, etro Styrene derivatives such as styrene, chloromethylstyrene, trifluorostyrene, trifluoromethylstyrene and aminostyrene: acrylonitrile derivatives such as acrylonitrile and α-acetoxyacrylonitrile: acrylic acid, methacrylic acid: methyl acrylate, lauryl acrylate, chloromethyl acrylate And acrylates such as ethyl acetoxyacrylate: cyclohexyl methacrylate, dimethylaminoethyl methacrylate, glycidyl methacrylate, tetrahydrofurf methacrylate Le, methacrylic acid esters such as hydroxyethyl methacrylate diethyl maleate,
Diethyl fumarate: vinyl ketones such as methyl vinyl ketone and ethyl isopropenyl ketone; vinylidene compounds such as vinylidene chloride, vinylidene bromide, and vinylidene cyanide: acrylamide, methacrylamide, N-butoxymethylacrylamide, N-phenylacrylamide;
Acrylamide derivatives such as diacetone acrylamide and N, N-dimethylaminoethyl acrylamide: fatty acid vinyl derivatives such as vinyl acetate, vinyl butyrate, and vinyl caprate: thiofatty acid derivatives such as phenyl thiomethacrylate, methyl thioacrylate, and vinyl thioacetate : Further N-
Vinyl succinimide, N-vinyl pyrrolidone, N-vinyl phthalimide, N-vinyl carbazole vinyl furan, 2-vinyl bend furan, vinyl thiophene, vinyl imidazole, methyl vinyl imidazole, vinyl pyrazole, vinyl oxazolidone, vinyl thiazole,
There are heterocyclic vinyl compounds such as vinyltetrazole, vinylpyridine, methylvinylpyridine, 2,4-dimethyl-6-vinyltriazine, and vinylquinoline.

The crosslinkable polymerizable monomer which is a constituent unit of the bilirubin adsorbent of the present invention includes divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene, divinylethylbenzene, divinylphenanthrene, trivinylbenzene, divinyldiphenyl, divinylphenyl. Ether, divinyl diphenyl sulfide,
Divinyldiphenylamine, divinylsulfone, divinylketone, divinylfuran, divinylpyridine, dipyrylquinoline, di (vinylpyridinoethyl) ethylenediamine, diallyl phthalate, diallyl maleate, diallyl fumarate, diallyl succinate, diallyl carbonate, diallyl oxalate, adipin Diallyl acid, diallyl sebacate, diallyl tartrate, diallylamine, triallylamine, triallyl phosphate, triallyl tricarballylate,
Triallyl aconitate, triallyl citrate, N, N
-Ethylenediacrylamide, N, N-ethylenedimethacrylamide, N, N-methylenedimethacrylamide,
Ethylene glycol methacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate,
1,3-butylene glycol diacrylate, 1,6-
Hexanediol diacrylate, trimethylpropane triacrylate, pentaerythritol tetraacrylate, triallyl isocyanurate, 1,2,5-triacryloylhexahydro-1,3,5-triazine, diallylmelamine and the like are included.

Among them, a styrene-based polymer and a copolymer with the above-mentioned vinyl compound are more preferably used in order to introduce a functional group which is chemically stable and easily. Further, those containing 70% by weight or more of the polymer of the styrene-based compound and the copolymer with the vinyl compound give more preferable results as the material used for the bilirubin adsorbent of the present invention. For example, in a styrene-divinylbenzene copolymer, styrene, ethylbenzene, divinylbenzene and toluene, octanol and AIBN are used.
By stirring under the coexistence of (azobisisobutyronitrile), spherical porous particles of about 50 μm to 1,000 μm can be produced. Also, particles having various particle diameters and pore diameters can be produced by radical polymerization in a suspension polymerization system.

In order to improve the affinity for blood, the surface of the bilirubin adsorbent is coated with a hydrophilic coating layer made of a polymer for controlling the adhesion of platelets to the surface of the bilirubin adsorbent which comes into contact with blood cells. You may have. The original purpose of the hydrophilic coating layer is to increase blood compatibility, and it is necessary to show the degree of blood compatibility, but it is difficult to obtain blood stably and there are differences between blood. , A common stable evaluation is difficult. The degree of hydrophilicity can be expressed simply. If you show the preferred range of the degree of hydrophilicity,
The contact angle at 25 ° C. of air bubbles on a sheet-like or film-like solid surface in water is 20 ° or more.

The hydrophilic coating layer is desirably a polymer to prevent peeling during use. To give specific examples of the hydrophilic coating layer, if a polymer unit is exemplified by a name as a monomer, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2
-Hydroxybutyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, methoxydiethylene glycol methacrylate,
Acrylic acid or methacrylic acid and its derivatives such as methoxytriethylene glycol methacrylate and glycerol monomethacrylate; methoxypolyethylene glycols such as methoxytriethylene glycol; styrene derivatives such as diethylaminoethylstyrene, hydroxystyrene and hydroxymethylstyrene; vinylamine and vinyl alcohol Any monomer 1 such as a monomer having a vinyl group such as a segmented polyurethane, a segmented polyester, a mono (2-methacryloyloxyethyl) acid phosphate, and a mono (2-acryloyloxyethyl) acid phosphate; Polymers containing more than one kind, such as block copolymers, graft copolymers, and monomers having a polyethylene oxide chain and other polymerizable monomers RAFT copolymer and the like.
In particular, the polymer preferably has a hydroxyl group. There is no particular limitation on the mode of bonding of the hydroxyl groups in the polymer.

The polymer may be a homopolymer of the above-mentioned polymer units, or may be a copolymer of two or more. Further, the polymer unit having a basic functional group is 0.1 to 2
It may be a copolymer containing 0% by weight and a polymer unit having a hydroxyl group. These polymers are linear polymers,
There is no particular relation to the polymerization form such as a graft polymer or a crosslinked polymer.

The polymer coating layer can be formed by a grafting method, a precipitation method,
It may be obtained by any method such as a coating method and a covalent bonding method using a functional group on the surface of the porous body. Among them, the coating method is particularly preferable in practical use because the production operation is easy.

The shape of the bilirubin adsorbent is spherical,
Granular, fibrous, hollow fiber, flat membrane, etc. are effectively used, but spherical or granular are most preferably used from the surface of plasma circulation during plasma circulation. Spherical or granular average particle diameters of 10 μm to 2500 μm are easy to use,
The range is preferably from 25 μm to 1,000 μm, and more preferably from 50 μm to 600 μm.

[0039]

According to the removal apparatus of the present invention, the dissociating agent added to the plasma efficiently reacts with albumin to release bilirubin, so that bilirubin can be adsorbed and removed much more efficiently than before, and the selectivity of adsorption can be improved. This is an apparatus which is also excellent in terms of economical use of the dissociating agent.

[0040]

Embodiment 1 Plasova BR filled with an adsorbent having a quaternary ammonium group on the surface as a bilirubin adsorbent 1
As shown in FIG. 2, the blood circuit was used as the adsorbent 2 of the dissociating agent using -350 and Prasorber N-350 filled with activated carbon as an adsorbent (both manufactured by Asahi Medical Co., Ltd.). The adsorbent having a quaternary ammonium group on the surface is obtained by a mercury intrusion method and has an average pore diameter of 137 ° and a total pore surface area of 3.1 m ² /
ml, surface area 1.8 m ² / ml with a pore diameter of 100 ° or less,
The surface area was 2.4 m ² / ml having a pore diameter of 200 ° or less, and the ratio of the surface area of pores having a pore diameter of 200 to 2,000 ° to the total pore surface area was 10.2%. Activated carbon is a mercury intrusion method and has an average pore diameter of 114 ° and a total pore surface area of 26.2 m.
² / ml, surface area of 16.3 m ² /
ml, surface area of 23.7 m ² / m with pore diameter of 200 ° or less
l, pore diameter 200 ° or more to total pore surface area 2,000
The ratio of the surface area of the pores of 0 ° or less was 9.4%. Hepatic failure patient plasma is flowed from the plasma reservoir 8 by the plasma pump 6 at a flow rate of 2
Flowed at 0 ml / min. Sodium benzoate as a dissociating agent was dissolved in physiological saline at a 1 molar concentration to prepare a dissociating solution (dissociation agent reservoir 3). The dissociation solution was continuously added to the plasma of the liver failure patient at about 0.4 ml / min by the pump 4 and mixed (13 in the figure) so that the ratio of the plasma to the dissociation solution was 50: 1. 100 minutes after the start of the plasma flow, plasma was collected as follows, and the concentrations of total bilirubin and sodium benzoate were measured. The total bilirubin concentration was measured using a bilirubin BII test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) by the alkali azobilirubin method. In addition, sodium benzoate was measured by gas chromatography.

Sampling position 1 immediately after addition (1 in the figure)
The plasma was collected at 0) and the total bilirubin and sodium benzoate concentrations were measured to be 22.4 mg / d.
1, 17.9 mM. Sodium benzoate was well mixed. Plasma was collected at sampling position 2 (11 in the figure) at the outlet of Plasova BR-350, and the concentrations of bilirubin and sodium benzoate were measured to be 7.3 mg / dl and 18.7 mM, respectively. The adsorption rate of bilirubin was as high as 67.4%, but no adsorption of sodium benzoate was observed. Placer N
Plasma was collected at sampling position 3 (12 in the figure) at the outlet of -350, and the concentrations of bilirubin and sodium benzoate were measured. The results were 6.6 mg / dl and 7 μM or less, respectively. Little adsorption of bilirubin was observed, and sodium benzoate was adsorbed very well.

[0042]

Example 2 A solution prepared by dissolving purified bilirubin (Wako Pure Chemical Industries, Ltd.) in bovine plasma using the bilirubin adsorbent of Plasova BR-350 of Example 1 was treated with sodium benzoate for 60 m.
After adding each concentration of M, 20 mM and 4 mM, 3 ml of this solution
The above-mentioned bilirubin adsorbent was immediately added thereto, mixed gently, and then shaken for 5 minutes to adsorb bilirubin. While the total bilirubin concentration in the plasma immediately after preparation was 17.6 mg / dl, sodium benzoate after reaction with the bilirubin adsorbent for 5 minutes was added with 60 mM, 20 mM, 4 mM and each of the plasma without addition of sodium benzoate. The total bilirubin concentration was
3.31 mg / dl, 5.85 mg / dl, 9.80 m
g / dl and 11.63 mg / dl, and the plasma to which sodium benzoate was added showed high adsorptivity at any concentration of sodium benzoate.

[0043]

Example 3 The same operation as in Example 2 was carried out using sodium thiocyanate as a dissociating agent. While the total bilirubin concentration in the plasma immediately after preparation was 17.6 mg / dl, sodium thiocyanate after the reaction with the bilirubin adsorbent for 5 minutes was added with 60 mM, 20 mM, 4 mM, and after addition of each plasma. , The total bilirubin concentration was 9.
44mg / dl, 10.71mg / dl, 11.00m
g / dl, a 11.63mg / dl, the plasma was added thiocyanate <br/> sodium showed higher adsorbing at any thiocyanate Na <br/> sodium concentration.

[0044]

Example 4 A styrene-divinylbenzene copolymer having a trimethylammonium group (Tokyo Organic Chemicals, total exchange capacity: 0.67 mEq / ml) was used as a bilirubin adsorbent. The physical properties of the bilirubin adsorbent, such as the pore diameter, its distribution, and the surface area, are determined using a mercury porosimeter (manufactured by Shimadzu Corporation, Micromeritics Poisizer 9).
320). The measurement results were obtained using a pore plot system (manufactured by Shimadzu Corporation, 9320-PC2 (V1.
0)). The average pore size of the bilirubin adsorbent is 6
57 °, volume-based mode diameter of 1,646 °, total pore surface area of 23.0 m ² / ml, total pore volume of 0.377 m
1 / ml. Further, the surface area of pores having a pore diameter of 100 mm or less is 5.0 m ² , and the pore surface area of 200 mm or less is 9.7 m ² , 200
The volume of pores having a pore diameter of 1,000 to 80,000 is not more than 12.1 m ² , the pore volume of not less than 100 to 1,000 is 0.165 ml / ml, and the pore volume of not less than 1,000 to 80,000 is not more than 0.12 ml. It was 176 ml / ml. At this time,
The ratio of the surface area of the pores having a pore diameter of 100 to 1,000 mm to the total pore surface area of 60 to 80,000 mm is 65.2%, and the pore diameter of 1,000 to the surface area of the pores having a pore diameter of 100 to 1,000 mm is 65.2%. 80,
The ratio of the surface area of the pores having a pore size of 100 to 1,000 mm to the total pore volume of the pores having a pore size of 60 to 80,000 is 40.0%.
The ratio of the volume of pores having a pore size of 1,000 to 80,000% to the total pore volume of 3.8% and pore sizes of 60 to 80,000% was 46.7%.

After dissolving bilirubin in a bovine albumin solution at a high pH in advance, a neutral pH solution was added to heparin (Novo Nordisk A / S, Novo Heparin,
1 ml of this heparin contains 1,000 units of heparin)
Bovine plasma containing 5 units / ml of
g / ml to prepare a test solution. After filling 1 ml of the bilirubin adsorbent into the mini column, the flow rate was 0.0
Priming was performed in the same manner as in practical use by flowing 5 ml of a physiological saline solution containing heparin (3 units / ml) at 6 ml / min. The test liquid prepared above was flowed at a flow rate of 0.06 ml / min through the primed bilirubin adsorbent-filled minicolumn. 8 ml of the effluent from the mini-column was collected, and the heparin concentration was measured.
53.1%, and the adsorption rate was as good as 70% or less. The effect of the dissociating agent was measured in the same manner as in Example 2 using this bilirubin adsorbent. While the total bilirubin concentration in the plasma immediately after preparation was 18.6 mg / dl, sodium benzoate after reaction with the bilirubin adsorbent for 5 minutes contained 60 mM, 20 mM, 4 mM and 4 mM of each plasma. The total bilirubin concentration was 1.17 respectively.
mg / dl, 2.93 gm / dl, 4.90 mg / d
1, 7.78 mg / dl, and the plasma to which sodium benzoate was added showed high adsorption at any concentration of sodium benzoate.

[0046]

Example 5 The styrene / divinylbenzene copolymer having a trimethylammonium group used in Example 4 was used as an adsorbent. The adsorbent was coated with polyhydroxyethyl methacrylate by immersing it in a 40% ethanol solution containing 0.5% polyhydroxyethyl methacrylate at a volume ratio of 1: 2. No clear change in pore size was observed before and after coating. Bilirubin was dissolved in bovine plasma in the same manner as in Example 4, and bovine blood cells obtained by centrifugation were mixed at 40 v / v% to obtain bilirubin blood. The blood is flowed over the coating adsorbent at a flow rate of 0.18 ml / min,
12 ml were collected. Otherwise, the effect of the dissociating agent was measured in the same manner as in Example 4. While the total bilirubin concentration in the plasma immediately after preparation was 16.1 mg / dl, the total bilirubin concentration in each of the collected blood with and without sodium benzoate added at 60 mM, 20 mM, 4 mM was 1. 33 mg / dl, 2.71 mg / dl, 4.3
It was 0 mg / dl and 6.73 mg / dl, and the blood to which sodium benzoate had been added showed high adsorptivity at any concentration of sodium benzoate.

[0047]

Comparative Example 1 In the same manner as in Example 1, patient plasma was refluxed without mixing with a dissociating agent, and the total bilirubin concentration was measured.
The bilirubin concentrations at sampling position 2 and sampling position 3 were 13.9 mg / dl, 12.
It was 5 mg / dl, and the bilirubin adsorbent had a bilirubin adsorption rate of 37.9%.

[0048]

Comparative Example 2 The activated carbon of bisorbin was measured in the same manner as in Example 2 except that activated carbon of Plasova N-350 of Example 1 was used as an adsorbent. Sodium benzoate was added to a solution in which purified bilirubin (Wako Pure Chemical Industries) was dissolved in bovine plasma at a concentration of 60 mM, 20 mM, and 4 mM. The activated carbon was immediately added to 3 ml of this solution, and the mixture was gently mixed. The mixture was shaken for 5 minutes to adsorb bilirubin. While the total bilirubin concentration in the plasma immediately after preparation was 17.6 mg / dl, the total bilirubin in each of the plasma was added with 60 mM, 20 mM, 4 mM and without sodium benzoate after reacting with activated carbon for 5 minutes. The concentration was 10.81 each
mg / dl, 11.20 mg / dl, 12.59 mg /
dl, 12.18 mg / dl, and the effect of adding sodium benzoate was hardly observed. At this time, sodium benzoate could not be detected in any of the samples. Since sodium benzoate was adsorbed on activated carbon immediately after the start of the reaction, it could not sufficiently react with the albumin / bilirubin complex, so that the effect of sodium benzoate was hardly obtained.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing one embodiment of the present invention.

FIG. 2 is an explanatory diagram of the first embodiment.

[Explanation of symbols]

 1. Bilirubin adsorbent 2. 2. Dissociation agent adsorbent Dissociation agent reservoir 4. 4. Dissociation agent mixing pump Plasma separator 6. Plasma pump 7. Blood pump 8. Plasma reservoir 9. Blood / plasma mixing unit 10. Sampling position 1 11. Sampling position 2 12. Sampling position 3 13. Plasma / dissociation agent mixing section

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshiro Ueda 215 Furuya, Wakayama 1 IM Hachimanmae 501 (56) References JP-A-54-482 (JP, A) JP-A-56-66262 (JP, A) JP-A-63-275351 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61M 1/36 530 B01J 20/26

Claims (1)

(57) [Claims] 1. A plasma reservoir (8) for plasma supply or plasma separation
Vessel (5), plasma pump (6) and non-activated carbon-based bilirubi
The removal means (1) and the dissociation agent removal means (2)
Plasma flow path arranged in this order from
Serum pump (6) and means for removing inactive carbon-based bilirubin
In the plasma channel between (1) and the dissociation agent reservoir (3),
Disassembly agent supply flow path with combined pump (4)
Removal of albumin binding bilirubin, characterized in that there
Equipment .