JPS6226073A - Method and apparatus for direct infusion and adsorption of blood - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to direct blood perfusion, which attempts to adsorb and remove components unnecessary for living organisms or malignant components from blood by direct perfusion of blood. This invention relates to an adsorption method and device.

More specifically, the present invention focuses on low-density riboproteins, which are thought to be closely related to various diseases caused by increased plasma lipids, cancer, immunoproliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, It is expressed in body fluids such as blood and plasma in diseases and phenomena related to the body's immune function such as allergies and rejection reactions during organ transplants, kidney diseases such as nephritis, and liver diseases such as hepatitis, and is the cause of the disease or A method of adsorbing and removing malignant substances from body fluids, which are thought to be closely related to disease progression, in particular by bringing blood into direct contact with an adsorbent material to adsorb malignant substances in the presence of blood cells. The present invention relates to a method and apparatus for doing so.

(Prior art) The main adsorbents that have been used or studied for the purpose of purifying body fluids include activated carbon used as an artificial liver for liver diseases, and materials coated with hydrophilic polymers. Activated charcoal, heparin-immobilized agarose used as a low-density lipoprotein adsorbent for familial hypercholesterolemia (Lupien, P-J, et.
8E: A new approach
t.

the management of family
l hypercholesterolemia.

Removal of plasma-choles
terol based on the principle
le of affinity chromatogr
aphy, Lancet.

2: 1261-1264.1976,), glass powder, glass peas (Carlson, L. 8: Chromatographicsepara.
tion of serum 1ipoprot
eins on glass powder col
ums, Description of the m
method and some application
s, Cl1n, Chim, Acta+ 5:
528-538.1960. ).

In addition, in recent years, special substances that the present inventors discovered as adsorbents for various autoantibodies and immune complexes have been chemically added to carriers that have biological and/or chemical selective interactions with the adsorbed substances. There are various adsorbents that are held together by bonding (Japanese Patent Application Laid-open No. 57-77624, Japanese Patent Application Laid-open No. 57-77625).
, JP-A-57-122875, JP-A-57-1341
64, Japanese Patent Publication No. 57-156035).

(Problem to be solved by the invention) Among these adsorbents, activated carbon is the only one that uses heparin as an anticoagulant and allows a large amount of blood to flow directly into a column filled with the adsorbent. In the case of highly hydrophilic adsorbents, platelet adhesion may occur, which may pose a problem. Furthermore, even in the case of activated carbon, although the adhesion of platelets is less than that of hydrophilic adsorbents, adhesion sometimes occurs, and improvement has been desired.

Such platelet adhesion problems can be improved by using sodium citrate or prostaglandin I2 as anticoagulants, but
In the case of sodium citrate, the large volume used can lead to dilution of the blood, and in the case of extracorporeal circulation therapy, the patient's blood volume increases, resulting in a condition called hypervolemia. In order to prevent this, water must be removed from the blood, and side effects such as numbness of the lips may occur due to large amounts of sodium citrate entering the body. In addition, prostaglandin
In case 2, a serious side effect of lowering the patient's blood pressure occurs during extracorporeal circulation treatment.

Therefore, there has been a desire for a method and device that can suppress platelet adhesion without side effects in direct perfusion adsorption of blood using heparin as an anticoagulant.

(Means for Solving the Problems) The present inventors have solved the above-mentioned problems of direct blood perfusion adsorption, and as a result of intensive research to provide a simple and safe method and device, We have discovered that by contacting blood with an adsorbent after adsorbing calcium, platelet adhesion can be surprisingly suppressed while the adsorbent's ability to adsorb malignant substances is maintained, and we have further achieved this goal. As a means for this, by bringing the blood into contact with a cation exchanger on the blood inlet side of the direct blood perfusion adsorption device, there is no change in blood volume, drug contamination, etc., and direct blood perfusion adsorption can be performed safely. We have discovered that this can be done, and have completed the present invention.

That is, the present invention is a direct blood perfusion adsorption method characterized in that blood is brought into contact with a cation exchanger and then brought into contact with an adsorbent. The blood introduction section, the cation exchange column, the adsorption column, and the blood extraction section are arranged in this order. This is a direct blood perfusion device featuring:

Blood as used in the present invention refers to a body fluid containing formed components such as red blood cells, white blood cells, and platelets.

The cation exchanger referred to in the present invention can be classified according to the manufacturing method (11, by copolymerizing a monomer and a crosslinking monomer to synthesize a small spherical basic polymer of a desired size, Next, (2) products in which anionic functional groups are chemically introduced, (2) products in which low molecular weight materials with anionic functional groups are made into polymers by condensation polymerization, or products in which anionic functional groups are created by chemically reacting the condensation polymers. (3) gels or inorganic porous materials such as cellulose, dextran, agarose, etc., with anionic functional organs chemically introduced onto the surface. Any water-insoluble body having an exchangeable anionic organ, capable of adsorbing calcium from blood, and having no adverse effect on body fluids or living organisms is sufficient.Commercially available cation exchange resins, chelate resins, etc. are preferable. used.

Shapes include fibrous, particulate, rod-like, hollow fiber, etc.
Various shapes can be used, but particles, especially spherical ones, are recommended because they cause less damage to blood cells. Furthermore, the larger the diameter of the particles, the smaller the surface area with which blood cells come into contact, which is preferable, but there is also a balance with the adsorption efficiency of calcium ions, and particles with a diameter of 0.03 to 5 mm are usually used. More preferably the range is 0.1 to 4 mm
The more preferable range is 0.2 to 3 mm.

Further, the internal physical structure of the cation exchanger is preferably porous in terms of adsorption efficiency. This makes it possible to increase the surface area for adsorption of calcium, resulting in good efficiency.

The pore size does not need to be very large as long as it allows calcium ions to pass through, and it is necessary to prevent the particle surface from becoming rough, adsorbing high molecular weight plasma proteins, activating coagulation, and activating the fibrinolytic complement system. Smaller is preferable.

In the case of a gel type cation exchanger, the pore size of the cation exchanger is usually expressed by the exclusion limit molecular weight measured using a virus or protein. Although it is possible to use 40 million or more, it is easier to use less than 4 million.
00,000 or less is more preferably used, and 30,000 or less is desirable. However, it is necessary to have a pore size larger than that which can accommodate calcium.

Furthermore, when the cation exchanger is a resin, the pore distribution is usually measured using a porosimeter using mercury intrusion, and is often expressed as an average pore diameter.

Although an average pore size of 3000 Å or more can be used, it is easy to use a pore size of 1000 Å or less, and 300 Å or less is more preferably used.

More preferably, the pore size is 100 Å or less, but the pore size must be larger than that for calcium ions.

The base material of the cation exchanger itself is preferably highly hydrophobic to some extent because this suppresses platelet adhesion.

As for the material, a material prepared by copolymerizing divinylbenzene with a monomer having a vinyl group, such as styrene or methacrylic acid, and into which an exchange group is introduced is preferably used. Needless to say, the base material is not limited to this, and may be inorganic, organic, natural, or synthetic.

Further, it is preferable that the cation exchanger does not deform under pressure due to blood flow, and it is preferable that the cation exchanger is hard.

Specifically, the cation exchanger has a diameter ↓Omm and a length of 50mm.
When filling a mm0 column and passing water through it, it is preferable that the volume shrinkage of the cation exchanger is 15% or less when the difference between the inlet pressure and the outlet pressure of the column is 200 mmHg. More preferably, it is 10% or less, more preferably 5% or less, and even more preferably 3% or less.

Anionic functional groups capable of exchanging cations include sulfonic acid groups, phosphoric acid groups, carboxyl groups, etc. Among them, weakly acidic carboxyl groups are preferably used. The anionic functional group may be a monoanion, but a polyanion is also preferably used.

The adsorbent used in the present invention is a water-insoluble substance that has a surface that can bind to an adsorbed substance, and the material surface itself may have adsorption properties, such as activated carbon, silica gel, and glass. Alternatively, it may be a carrier that does not show much adsorptive property itself and has a ligand capable of binding to the adsorbed substance immobilized thereon, and has a surface that can biochemically, physically, or chemically bind to the adsorbed substance. refers to something that exists.

Examples of ligands that can bind to adsorbed substances include heparin, sulfated polysaccharides such as dextran sulfate,
Alternatively, anti-low-density riboprotein antibodies, synthetic polyanions, etc. may be mentioned.

For the treatment of systemic lupus erythematosus, homopolymers or copolymers of mono-, di-, and trinucleotides such as adenine, guanine, cytosine, uracil, and thymine, and naturally occurring DNA are used for adsorption and removal of anti-nuclear antibodies and anti-DNA antibodies. , RNA, and other nucleic acids. In addition, for the adsorption and removal of DNA, RNA, and ENA present in the blood, anti-Ippont* DNA antibodies, anti-two-chain DNA antibodies, and anti-RNA are available.
Examples include antibodies, anti-nucleic acid antibodies such as anti-ENA antibodies, and basic compounds such as methylated albumin actinomycin D.

Furthermore, for adsorption and removal of immune complexes in blood, there may be mentioned complement components such as ctq, specific proteins such as protein A, and antibodies against immune complexes such as anti-heavy chain constant region 2 phase antibody.

For the treatment of chronic rheumatoid arthritis and malignant rheumatoid arthritis,
Modified γ-globulin, denatured by chemical denaturation (aggregation) methods such as urea, guanidine hydrochloride, mercaptoethanol, surfactants, and organic solvents, and physical denaturation (aggregation) methods such as heat, ultrasound, and gas bubbling. Antigen-like substances for rheumatoid factors such as immunoglobulin, aggregated γ-globulin, aggregated immunoglobulin, Fc region of immunoglobulin, heavy chain constant phase 2 of immunoglobulin, and modified products thereof by the above-mentioned modification method, and anti-rheumatoid factor. Examples include antibodies. In addition, for removing immune complexes from rheumatism, antibodies against immune complexes such as complement components such as C1q, specific proteins such as protein A, and anti-heavy chain constant region phase 2 antibodies may be mentioned.

For the treatment of Hashimoto's disease, thyroglobulin, a microsomal fraction of the thyroid, is used, and for the treatment of myasthenia gravis,
Examples include neuromuscular acetylcholine receptor fraction components.

Glomerular basement membrane components are used to treat glomerulonephritis, platelet membrane components and platelet granule fraction components are used to treat idiopathic thrombocytopenic purpura, and transcortisin and anti-cortisin antibodies are used to treat Cushing's syndrome. Can be mentioned.

For the prevention and treatment of hepatitis, antibodies against surface antigens of viruses such as hepatitis A virus and hepatitis B virus may be used, and for the treatment of hypertension, anti-angiotensin antibodies may be used.

Anti-lymphocyte antibodies such as anti-B cell antibodies, anti-pressor T antibodies, and anti-lymphocyte antibodies are used to treat immune diseases based on lymphocyte abnormalities, and protein A and anti-lymphocyte antibodies are used to treat cancers such as breast cancer. Examples include immunoglobulin antibodies.

Ligands that can be used in the present invention are not limited to the above examples, but include lectins such as congnitinin and concanavalin A1 phytohemaagglutinin, nucleic acids,
Known substances capable of binding to adsorbed substances such as amino acids, lipids, protamines, antigens, antibodies, enzymes, substrates, and coenzymes can be used.

It is also possible to use a carrier holding two or more types of glue gants. Furthermore, two or more types of carriers holding ligands can be used in combination.

The carrier needs to be a fibrous or rod-like porous body, and it must contain an activated functional group (a nucleophilic reactive group having active hydrogen such as a hydroxyl group, an amino group, a carboxyl group, a thiol group, a silanol group, etc.) It is preferable to have this on the surface.

All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, and insolubilization by precipitation on the surface of a polymer can be used to bond the ligand to the carrier. It is preferable to use it after fixation and insolubilization.

Therefore, commonly known methods of activating immobilized enzymes, carriers used in affinity chromatography, and binding methods of ligands can be used.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, 1,1'-carbonylbiimidazole method, and silane coupling agent. Examples include activation methods.

The activation method of the present invention includes an amino group, a hydroxyl group,
It is not limited to the above examples as long as it can undergo a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as a carboxyl group, a thiol group, or a silanol group.

In the case of an inorganic porous carrier, a silane coupling agent is preferably used.

Next, the direct blood perfusion adsorption device of the present invention will be explained.

FIG. 1 is a schematic diagram showing an example of the direct blood perfusion device of the present invention. Blood is introduced through a blood introduction section 1 and introduced into a cation exchange column 2, where calcium is adsorbed and removed. The blood from which calcium has been removed is then introduced into an adsorption column 3, where malignant substances are adsorbed and removed. Since calcium has been removed in the cation exchange column 2, platelet adhesion is unlikely to occur in the adsorption column 3. There is no clogging caused by blood cells within the chamber. The blood purified by the adsorption column 3 is then taken out from the blood outlet 4 and returned to the patient in the case of extracorporeal circulation treatment. Furthermore, the components removed by the cation exchange column 2 can be replenished near the blood outlet, if necessary.

The amount of ion exchanger in the cation exchange column 2 is determined by the amount of blood to be treated and the exchange capacity of the ion exchanger, but it is sufficient as long as it can adsorb an effective amount of calcium ions in the blood to be treated.

Further, the ion exchange column 2 and the adsorption column 3 may be integrated, as long as their positional relationship is not reversed.

The blood introduction section 1 and the blood outlet section 4 are circuits for introducing and discharging blood, and usually a vinyl chloride tube, a silicone tube, or the like is used.

(Effects of the Invention) By using the direct blood perfusion adsorption method and adsorption device of the present invention, direct perfusion of blood to the adsorbent, which was previously impossible, has become possible, allowing separation of blood into blood cells and plasma. There is no longer a need for a blood vessel, the volume of blood drawn out of the body during extracorporeal circulation is reduced, and the blood flow rate can also be reduced, making it much easier to secure blood vessels.

Furthermore, since calcium is first removed from the blood using a cation exchange resin, platelet loss is greatly reduced.
It has become a safe treatment system for repeated extracorporeal circulation.

The present invention is applicable to a general method of purifying and regenerating autologous blood, and is applicable to various diseases caused by increased serum lipids such as familial hyperlipidemia, cancer, immunoproliferative syndrome, and rheumatoid arthritis. , collagen diseases such as systemic lupus erythematosus, autoimmune diseases such as myasthenia gravis, allergies, diseases and phenomena related to the body's immune function such as organ transplant rejection, kidney diseases such as nephritis, and liver diseases such as hepatitis. It can be effectively used for extracorporeal circulation treatment of diseases.

(Example) Example 1 Using the experimental circuit shown in FIG. 2, an experiment was conducted to adsorb and remove low-density lipoprotein from the blood of a patient with heparin-added familial hypercholesterolemia.

Cation exchange column 2 has an inner diameter of 8 m111, a length of 51 m,
DIAIONWK Na-type Nuccryl weakly acidic cation exchange resin in a cylindrical container with an internal volume of 0.25 m It
II (manufactured by Mitsubishi Chemical Industries, Ltd.) filled with 0.25 m It was used.

The DIAION WK 11 used was white and opaque spherical, and the particle size was reduced to 0.5 by sieving.
A diameter range of 9 to 0.84 μ was used, and the cation exchange capacity was 3.1 meq/ml. Although the pore size was below the measurement limit of the mercury porosimeter (40 people), the pore size was large enough to adsorb calcium.

The adsorption column 3 uses a hydrophilic carrier with polyacrylic acid immobilized as an adsorbent, and 1 ml of this is used as an adsorbent with an inner diameter of 8 mm.
It was used by filling a column with a length of 20 m+n.

The adsorbent was prepared as follows.

Hinyl acetate 100g, triallyl isocyanurate 41
．． 4g (X=0.40), ethyl acetate 100g
, heptane 100 g, polyvinyl acetate (polymerization degree 50
0) A homogeneous mixture of 7.5 g and 3.8 g of 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, and 0% sodium dihydrogen phosphate dihydrate.
．． 05% by weight and 1.5% by weight of disodium hydrogen phosphate dodecahydrate dissolved in water 400n/! After stirring thoroughly, the mixture was heated at 65°C for 18 hours, and then for 75 hours.
Suspension polymerization was carried out by heating and stirring at °C for 5 hours to obtain a granular copolymer. After filtering, washing with water, and then extracting with acetone, 46.5 g of caustic soda and 21 g of methanol were added.
The transesterification reaction of the copolymer was carried out at 0° C. for 18 hours.

The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit (q O1+ ) per unit weight was 9. Ome
q/g, specific surface area is 60n? /g, and the exclusion limit molecular weight with dextran was 6×105.

Next, 10 g (dry weight) of the obtained gel was suspended in 120 ml of dimethyl sulfoxide, and 78.3 ml of epichlorohydrin and 10 ml of 50% sodium hydroxide were added to the suspension.
ml was added, and the activation reaction was carried out with stirring at 30° C. for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. The amount of epoxy group bonded in the activated gel is 110. dma! ! Met.

Using the epoxy group-bonded gel, polyacrylic acid (weight average molecular weight 9×10', manufactured by Altrift, USA) was bound as a ligand to prepare an adsorbent.

Dilute 4.5 g of polyacrylic acid to 500 ml with distilled water,
This was used as a ligand solution. The epoxy group-binding gel was added to this ligand solution, and the ligand binding reaction was carried out at 50° C. for 16 hours with shaking.

Thereafter, it was thoroughly washed with water and converted into Na type to obtain a low-density riboprotein adsorbent.

In the figure, 6 is heparin at 5U/m! ! The blood of a patient with familial hypercholesterolemia was added at a ratio of 1,000 to 1,000 ml, and the mixture was placed in a polyethylene beaker 5 and gently shaken.

Blood 6 is sent to a cation exchange column 2 by a peristaltic pump 7, where calcium is removed, and then sent to an adsorption column 3, where low-density riboproteins are adsorbed. It was done. The blood flow rate was 0.3 m It /min, and the pump was stopped when the blood 9 in the polyethylene beaker reached 20 mf.

During the experiment, there was almost no fluctuation in the flow rate, and the pressure in the circuit between the pump 7 and the cation exchange column 2 was 20 mmHg.
It was below.

The platelet concentration of blood 9 after passing through the column is 2. OX10S
/mm”, and the platelet concentration before passing through the column is 2.2X
There was no significant difference from IO' 7mmff, and there was little adhesion of platelets to the column.

In addition, the low-density riboprotein concentration of blood 9 after passing through the column is 220 mg/a, and 670 mg/d before passing through the column.
It decreased to 33% of l.

Example 2 The same procedure as Example 1 was carried out, except that as the adsorbent packed in the adsorption column 3, a phenylalanine immobilized on a woody carrier was used, and as the blood, heparinized blood of a rheumatoid arthritis patient was used. A similar experiment was conducted to adsorb and remove rheumatoid arthritis prisoners from the blood.

As the adsorbent, the same carrier as in Example 1 was activated in the same manner and phenylalanine was immobilized thereon.

The immobilization conditions for phenylalanine are activated gel 100m
Phenylalanine 151IIIIOR for It
150ml of 0.1M carbonate buffer y-(pH 9,8) solution containing
The reaction was allowed to take place while stirring for hours. After this, it was thoroughly washed with water and used.

When blood was flowed in the same manner as in Example 1, there was almost no variation in flow rate during the experiment, and pump 7 and cation exchange column 2
The pressure in the circuit between the two was as low as 25mmt1g.

The platelet count was 2.8X10'/mm' before passing through the column, whereas it was 2.5X10'/mm' even after passing through the column.
-l+3, which did not decrease much.

Furthermore, the rheumatoid factor (measured using RAHA titer, a measurement kit manufactured by Fujirebio) was 640 before passing through the column, but it had decreased to 160 after passing through the column.

Comparative Example 1 An experiment was carried out in the same manner as in Example 2, except that the cation exchange column 2 was not used in Example 2.

When blood was allowed to flow, the flow rate tended to decrease over time, and platelet adhesion within the adsorption column was observed.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of the direct blood perfusion adsorption device of the present invention, and FIG. 2 is a schematic diagram of the experimental device used in the examples. 1--------------------------------------------------------------------------------Adsorption power ram 4------------------------------------ --- BEaker 6 .9------One Blood 7・-------Pump No. 7n Year 2n

Claims (2)

[Claims] (1) A direct blood perfusion adsorption method characterized by bringing blood into contact with a cation exchanger and then contacting an adsorbent. (2) Consists of at least a blood introduction part, a blood extraction part, a cation exchange column filled with a cation exchanger, and an adsorption column filled with an adsorbent, and includes a blood introduction part, a cation exchange column, an adsorption column, and a blood extraction part. A direct blood perfusion adsorption device characterized in that the parts are provided in this order.