JPS60241450A - Adsorbing material for purifying body fluids - 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 relates to a porous adsorbent having a surface capable of bonding with an adsorbed substance. More specifically, we will focus on low-density riboproteins, which are thought to be closely related to various vertebral diseases caused by increased plasma lipids, as well as cancer, immunoproliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, allergies, and organ transplantation. Diseases and phenomena related to the body's immune function such as rejection reactions, kidney diseases such as nephritis, liver diseases such as hepatitis, etc., are expressed in body fluids such as blood and plasma, and are closely related to the cause or progression of the disease. This invention relates to an adsorbent that adsorbs and removes malignant substances thought to be associated with body fluids.

(Prior art) The main adsorbents that have been used or studied for this purpose are, for example, activated carbon used as an artificial liver for liver diseases and hydrophilic polymers. Coated activated charcoal, heparin-immobilized agarose used as a low-density riboprotein adsorbent for familial hypercholesterolemia (Lupien, P-J, et al.
l: A new approach.

the management of family
l hypercholesterolemia.

Removal of plasma-cholest
erol based on the principle
e of affinity chromatogra
phy, Lancet.

221261-1264.1976,), glass powder, glass peas (Carlson, L, A,
:Chromatographic 5eparati
on of serum lipoproteins o
n glass powder columns+Des
cription of the method
d some applications.

Cl1n, Chim, Acta, 5: 528~
538, 1960. ). In recent years, the present inventors have discovered that special substances that have selective biological and/or chemical interactions with the adsorbed substances have been developed as adsorbents for various autoantibodies and immune complexes. There are various adsorbents that are held together by chemical bonds. (% Gansho 55--
128185, 128184.56-7152°189
23.41700) (Problems to be Solved by the Invention) Some conventional adsorbents do not have sufficient adsorption capacity as adsorbents for body fluid purification, and when used as an extracorporeal circulation therapy device, the adsorption capacity is insufficient. If a large amount of adsorbent is not used, a sufficient clinical effect cannot be achieved, and if a large amount of adsorbent is used, the priming volume may increase, resulting in undesirable effects on the patient. Improvements were desired. In addition, in order to create a compact and easy-to-operate adsorption column with a smaller priming volume, we also added traps.
It has been desired to increase the adsorption capacity of adsorbents.

(Means for Solving the Problem) The present inventors have solved the problems of conventional adsorbents as described above, and further, in order to make the adsorbent even more capable, As a result of intensive studies focusing on the structure of the adsorbent, we found that conventionally, pores with a diameter slightly larger than the diameter of the adsorbed substance exist with a sharp pore size distribution, but by increasing the adsorption surface area, high adsorption capacity can be achieved. However, it is believed that adsorbents with pores distributed over a wide range of pore sizes, from around the diameter of the adsorbed substance to several tens of times the diameter of the adsorbed substance, are highly effective. The inventors have discovered that the higher the adsorption capacity, the greater the full potential of the adsorption capacity, and have thus obtained the present invention.

That is, in the present invention, in a porous adsorbent having a surface capable of bonding with an adsorbed substance, where d is the diameter of the adsorbed substance, 70% or more of the total pore volume of the adsorbent has a pore diameter of 0.
It is distributed in the range of 2d to 50d, and the pore diameter '(5D) is 0.8D to 1.2 at any pore diameter.
This is an adsorbent for body fluid purification characterized in that the pore volume in the range D is less than 80 cm of the total pore volume.

Here, the porous adsorbent having gold on the surface that can bind to the adsorbed substance may be a porous material such as activated carbon, silica gel, glass, etc., which has adsorption properties on its surface; Gant may be immobilized on a porous carrier that does not show much adsorptivity and is capable of binding the adsorbed substance.
It is preferable to use an adsorbent that is a porous body with a sponge-like network structure that extends into the interior of the adsorbent, and has a surface that can biochemically, physically, and chemically bond with the substance to be adsorbed.

The diameter d of the adsorbed substance refers to the length of the axis of rotation (long axis) when the molecule is considered to be an elongated spheroid, and is defined by the following formula fl).

Here, M is the molecular weight determined from the measurement of any two of sedimentation velocity, diffusion, and viscosity, ■ is the partial specific volume of the solute, NVi Avogadro's number, and P is the axial ratio when the molecule is considered to be an elongated spheroid. (Length of minor axis)/(Length of major axis)] and is calculated from the friction ratio (Protein Chemistry 2, pp. 357-56
3. Edited by Mizushima Sanbu and Akahori Shibu, Kyoritsu Publishing).

The total pore volume and pore diameter are calculated using the mercury intrusion method (for example, 4. Catalyst Measurement Method, edited by the Catalysis Society, Jiheshokan, pp. 69 to 7).
This refers to the value calculated from the large mercury pressure curve obtained from page 3).

Here, the total pore volume is preferably 1 ltO.5 cc/f (dry adsorbent weight) or more, and more preferably 1.00071 or more. Preferably 2. It is more desirable that it is greater than OCC/f, and it is more desirable that it be greater than s, oct7y. If the material of the adsorbent is the same, the larger the value of the pore capacity, the larger the internal space volume of the adsorbent per unit volume, and the adsorption capacity of the adsorbed substance can be increased accordingly.

The pore size distribution of the adsorbent is preferably such that 70% or more of the total pore volume is within the range of 0.2 d to 50 d, when the diameter of the substance to be adsorbed is 1rd. That is, it is preferable that the particles are widely distributed on the pore diameter side larger than the diameter d of the adsorbed substance. Here, a range smaller than d, ie, 0.2d to 1. The inclusion of a pore size range of Od is due to the fact that the diameter d of the adsorbed substance is defined by the long axis of the elongated spheroid.

The distribution of pore diameters indicates that, when the pore diameter is iD, the pore volume in the range of 0.8D to 162D is the total pore size at any pore diameter (even when considering pore diameters from 0.2D to 50D). It is necessary that the pore volume be less than 80 inches. That is, it is preferable that the pores be concentrated only in a specific pore size range, or that the pores be distributed over a wide pore size range.

When trying to completely adsorb a certain substance II from blood or body fluids, K is:
It is desirable that the pores be concentrated in a pore size range similar to the diameter d of the adsorbed substance, but if the pore size distribution is narrow, the surface of the adsorbent particles may become clogged with coexisting substances that have a diameter larger than that of the adsorbed substance. I often get excited. In order to prevent clogging, it is possible to use an adsorbent with a large pore size, but in this case, the surface area of the adsorbent becomes small and the adsorption capacity of the adsorbed substance becomes low. . In the case of an adsorbent having such a narrow pore size distribution, it is extremely susceptible to the effects of coexisting substances in blood and body fluids, and it is extremely difficult to improve its adsorption performance.

On the other hand, in the case of adsorbents with a wide pore size distribution, coexisting substances with a diameter larger than that of the adsorbed substance are captured in the pores with a large pore size. It is thought that the book is less likely to be crushed, and as a result, the adsorption capacity can be significantly increased.

A more preferable pore size distribution is such that, at any pore size, the pore volume in the range of 0.8D to 1.2D is 75% or more of the total pore volume, preferably 70%. Below, the Vi of the mold is 65% or less.

Among adsorbents with a wide pore tt distribution, the pore size distribution is 0.2d.
When there is a certain degree of bias between Sod and Sod, it is preferable that the pressure is biased toward the small hole diameter side rather than biased toward the large hole diameter side.

Preferably, the pore size range from 0.2d to 5d contains 50 to 90% of the pore volume between 0.2d and 50d;
It is more preferable to include 5 to 85 inches, and preferably 60 to 80 inches.

Here, a porous adsorbent having a surface capable of bonding with an adsorbed substance will be exemplified. Examples of materials whose surface itself has adsorption properties include medium- and low-molecular-weight drugs for artificial livers,
Activated carbon is used to adsorb toxic substances, and porous glass is used to adsorb and remove cholesterol in the treatment of familial hypercholesterolemia. In addition, an example of a type of ligand immobilized on a carrier is a ligand for adsorption of low-density lipoprotein for treatment of familial hypercholesterolemia.
Examples include heparin or anti-low density riboprotein antibodies.

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. Examples include nucleic acids such as %RNA. My friend, DNA exists in the blood.

For adsorption and removal of RNA and gNA, anti-single-stranded DNA antibody,
Examples include anti-nucleic acid antibodies such as anti-double-stranded DNA antibodies, anti-RNA antibodies, and 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 C1q, specific proteins such as protein A, and antibodies against immune complexes such as anti-heavy chain constant region 2 phase antibodies.

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

For the treatment of Hashimoto's disease, KFi, thyroglobulin, and thyroid microluminous fraction components are listed, and for the treatment of myasthenia gravis, neuromuscular acetylcholine receptor fraction components are listed.

For the treatment of glomerulonephritis, glomerular basement membrane components, iCF'i for the treatment of idiopathic thrombocytopenic purpura, platelet membrane components, platelet granule fraction components, and for the treatment of Cushing's syndrome, transcortisin and anti-cortiscin antibodies. can be given.

For prevention and treatment of hepatitis, antibodies against surface antigens of viruses such as hepatitis A virus and hepatitis B virus can be mentioned.

Anti-angiotensin antibodies are used to treat hypertension.

For the treatment of immune diseases based on lymphocyte abnormalities, anti-lymphocyte antibodies such as anti-B cell antibodies, anti-pressor T antibodies, and pile helper T antibodies can be used.

Protein A and anti-immunoglobulin antibodies are used to treat cancer such as breast cancer.

Ligands that can be used in the present invention are not limited to the above examples, but include lectins such as congnitinin, concanavalin A5 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.

Examples of the carrier include organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, and vinyl polymer gel, which can be used in ordinary affinity chromatography. All of the materials for the carrier that can be used can be used, but they must meet the conditions of pore size and pore size distribution described above.

Examples of methods for binding a ligand to a carrier include covalent bonding, ionic bonding, physical adsorption, embedding, and precipitation insolubilization on the surface of a polymer. It is preferable to use it after being fixed or insolubilized. For this purpose, examples include commonly known methods for activating immobilized enzymes, carriers used in affinity chromatography, and methods for binding ligands.

Examples of activation methods include a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a halogenated triazine method, a bromoacetyl bromide method, an ethyl chloroformate method, a 1,1'-carbonyldiimidazole method, etc. can. The activation method of the present invention is not limited to the above examples as long as it can perform a substitution and/or addition reaction with a nuclear reactive group having active hydrogen such as a 7-mino group, hydroxyl group, carboxyl group, or thiol group of a ligand. .

In the case of an inorganic porous carrier, preferred examples include silane coupling agents such as r-glycidoxypropyltrimethoxysilane, γ-aminopropyltriethoxysilane, r-mercaptopropyltrimethoxysilane, and vinyltrichlorosilane. can.

The body fluid purifying adsorbent of the present invention is generally used by filling and maintaining the internal pressure of a container equipped with all the inlet and outlet ports for body fluid.

In the drawings, reference numeral 1 shows an example of an adsorption device containing the adsorbent for body fluid purification of the present invention, in which body fluid is introduced into an opening at one end of a cylinder 2 through a backing 4 with a filter 5 stretched inside. A cap 6 having an opening 5 is screwed into the opening at the other end of the cylinder 2, and a cap 8 having a body fluid discharge lower part is screwed into the opening at the other end of the cylinder 2 through a backing 4' having a filter 3' stretched therein, thereby forming a container. However, the gap between the filters 3 and 5' is filled with adsorbent and retained therein, thereby forming the entire adsorbent layer 9.

The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. When used for extracorporeal circulation, the appropriate volume of the adsorbent layer 9 is about 50-400.

When the device of the present invention is used for extracorporeal circulation, there are two methods as follows: One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or model plasma separator, and then pass the plasma components through the device to purify them and separate them into blood cells. One method is to return the blood to the body together with its components, and the other method is to pass blood taken from the body directly through a nuclear device and purify it.

(9) Regarding the passage rate of blood or plasma, since the adsorption efficiency of the adsorbent is very high, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be adjusted. A high passing speed can be provided regardless of the situation. Therefore, a large amount of body fluid can be treated.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

(Effects of the Invention) As described above, the adsorbent of the present invention has an extremely high adsorption capacity compared to conventional ones, so when used as an adsorbent for body fluid purification, the priming volume of the adsorption column can be reduced. Therefore, the burden placed on the patient due to the removal of blood from the body can be extremely reduced, and the column is also compact and easy to operate.

The adsorbent of the present invention can be applied to general uses for purifying and regenerating body fluids such as autologous plasma and blood, and can be used to treat various diseases caused by increased serum lipids such as familial hyperlipidemia, and cancer. ,
Immune proliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, etc., autoimmune diseases such as myasthenia gravis,
It can be effectively used for extracorporeal circulation treatment of 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.

(Example) Example 1 Using porous glass as an adsorbent, low-density riboprotein (diameter d = 25
0X) adsorption properties were investigated.

The porous glass used had a pore diameter of 50 to 12,500X (
The total pore volume (2,28
CC/fi') (095% distribution, 50-1250
When the pore size is D in the pore size range of 0.0 x, the maximum value in the pore volume range of 0.8D to 1.2D was 72cm, which is the total pore volume.

The above porous glass is CPG 500 (Electro
Made by Nucleonics, average pore size 515X) 10- is immersed in 0.5 N% NaOH260-
After performing a dissolution operation for 5 hours, the solution was thoroughly washed with water and dried. The pore distribution was measured using a mercury intrusion porosimeter manufactured by Kalkuchi Erba (Italy).

An adsorption experiment was conducted in which the above porous glass was immersed in heparin-added familial hypercholesterolemia patient plasma 12- and incubated at 57C for 3 hours with shaking. After this, the adsorbent was allowed to settle and the supernatant was analyzed and used and compared with the next patient's plasma.

In the analysis, low-density riboprotein (hereinafter abbreviated as LDL) was measured by turbidimetry.

As a result of the analysis, the LDL in the patient's plasma was 730 lR97d.
1, but after adsorption it decreased by 150~/dlK. That is, 69.6#+90 low-density riboproteins were adsorbed per 1 d of adsorbent.

Comparative Example 1 CPG500 (manufactured by Electro Nucleonics, average pore diameter 51sX) was used as the adsorbent except for the holes.
An experiment was conducted in the same manner as in Example 1.

CPG500Fi, pore size 50~12500X (0,
The total pore volume (1,02a;
/f/), 98 chis are distributed, but 50 to 12,500
When the pore diameter is D in the pore diameter range of A, 0.8D to 1.
The maximum value of the pore volume in the 2D range is 95% of the total pore volume.
%Met. That is, the pore size distribution was very sharp.

As a result of the adsorption experiment, the LDL in the patient's plasma was 730~/dj, whereas it was only 45041/dj after adsorption. That is, 541 per adsorbent ITat
Only n9 LDL was adsorbed.

Example 2 Using porous glass as an adsorbent, low-density riboprotein (diameter d = 25
0X) adsorption properties were investigated.

The porous glass used had a pore diameter of 50 to 12,500X (
The total pore volume (4,38
80% of CC/7) is distributed, 50-12500
When the pore diameter is D in the pore diameter range of 0.8D to 1.2
The maximum value of the pore volume in the range D is 20% of the total pore volume.
Met.

The above porous glass is CPG500 (manufactured by Electro Nutaleonics, average pore diameter 493X) + OsI
/i 1 N, immersed in NaOH260-, 30p
After performing a dissolution operation for 15 hours, the solution was thoroughly washed with water and dried.

An adsorption experiment was conducted in the same manner as in Example 1, and while the LDL in the patient's plasma was 730 mg/dt, it decreased to 170~/dl after adsorption. That is, adsorbent 1
~67.2 low density riboproteins were adsorbed per rnl.

Example 3 Using porous glass as an adsorbent, low-density riboproteins (diameter d==2
50^) Adsorption properties were investigated.

The porous glass used had a pore diameter of 50 to 12,500 (0
, 2d to 50d) with a total pore volume (3,07Q
C/9) is distributed, and when the pore size iD is in the pore size range of 50 to 12,500^, the maximum value in the pore volume range of 0.8D to 1.2D is 60% of the total pore volume. %Met.

The above porous glass is CPG350 (manufactured by Electro Nutaleonics, average pore diameter 345λ) 10 ml
Soaked in f5N% NaOH50-2 at room temperature.
After performing the dissolution operation for several hours, the product was thoroughly washed with water and dried.

When an adsorption experiment was carried out in the same manner as in Example 1, the LDL in the patient's plasma was 750 Da/a, but after adsorption, it decreased to 130~/a. That is, 72 mg of low-density riboprotein was adsorbed per 1 inch of adsorbent.

Comparative Example 2 An experiment was conducted in the same manner as in Example 1, except that CPG550 (manufactured by Electro Nucleonics, average pore size 5 as 5.) was used as the adsorbent.

cpa35oVi, pore size 50~12500^(0,2d
Total pore volume (1,02α/7) in the pore size range of ~50d)
However, when considering the entire pore size in the pore size range of 50 to 12,500 x, the maximum value in the pore volume range of 0.8D to 1゜2D is 95 of the total pore volume. It was Chi. That is, the pore size distribution was very sharp.

As a result of the adsorption experiment, the LDL in the patient's plasma was 750 η/a, but after adsorption it was only 640/di. That is, 10.81R per adsorbent
Only LDL of Ii was adsorbed.

Comparative Example 3 An experiment was carried out in the same manner as in Example 1, except that CPG1400 (manufactured by Electro Nucleonics, average pore diameter 1489 λ) kumquat was used as the adsorbent.

CPG 1400d, pore diameter 50~12500A (0
, 2d to 50d) with a total pore volume (1,02c
c/2) is distributed, but 50 to 12500
When the pore diameter is D in the pore diameter range of ^, 0.8D to 1.2
The maximum value of the pore volume in the range D was 94 cm, which is the total pore volume. That is, the pore size distribution was very sharp.

As a result of the adsorption experiment, the LDL in the patient's plasma was 730IRQ.
/#, but after adsorption it only decreased to 480η/a. That is, 30 per adsorbent
Only 1RG/LDL was adsorbed.

Example 4 Porous glass surface 1 using silane coupling agent
c) Adsorption of anti-acetylcholine receptor antibody (immunoglobulin G, diameter d==300X) in plasma of myasthenia gravis subjects was investigated using an adsorbent bound with IJ gutophane.

The adsorbent used had a pore diameter of 60 to 15000λ (0.2d
85% of the total pore volume (2,50 cc/liter) is distributed in the pore size range of ~50d), and when the pore size is D in the pore size range of 6o~tsooo The maximum value of pore volume was 63% of the total pore volume.

The above adsorbent was obtained as follows.

CPG75 (manufactured by Electro Nutaleonics).

Average pore diameter 79λ) 20d (i70.5N, NaOH52
The sample was immersed in 0i water and dissolved at 20c for 8 hours, then thoroughly washed with water and dried. After washing 5ynl of this porous glass with acetone, it was immersed in a solution of 20% v/v, r-glyndoxybrobyltrimethoxysilane in acetone, and reacted at 50C for 40 hours without shaking. I let it happen. The resulting activated porous glass total acetone, water, 0.1
After sequential washing with M sodium carbonate buffer (pH 9,8), 10 mK$L in 0.1 M sodium carbonate buffer containing tryptophan of 81.5 to 16 at 50C.
The immobilization reaction was carried out with stirring for hours. Thereafter, it was thoroughly washed with water to obtain an adsorbent for body fluid purification. The amount of tryptophan immobilized on the porous glass was 55 μmol/−.

In the adsorption experiment, 1 d of the obtained adsorbent was soaked in 3 layers of myasthenia gravis patient plasma and heated at 37 C for 6 hours without shaking.
This was done by incubating for hours. After incubation, the adsorbent was sedimented and the entire supernatant was analyzed and compared to the patient plasma used.

In the analysis, anti-acetylcholine receptor antibody was
It was measured by the gG method.

As a result, while the anti-acetylcholine receptor antibody in the patient's plasma was 23 cmot/me, Fi'pmot/rnlK decreased after adsorption.

Comparative Example 4 Activation with silane and fixation of tryptophan were performed in the same manner as in Example 4, except that CPG75 (average pore size 79X) was used as the porous glass without being dissolved in NaOH. The amount of immobilized tryptophan is 38μmot
It was /-.

This adsorbent has a pore size of 60 to 1 sooo
Total pore volume (0,40cc/r) in the pore size range of 50d)
However, when the pore size is D in the pore size range of 60 to 15,000λ, the maximum value in the pore volume range of 0.8D to 1.2D is 90% of the total pore volume. Met. That is, the pore size distribution was very sharp.

An adsorption experiment was conducted in the same manner as in Example 4, and it was found that 25 p m of anti-acetylcholine receptor antibodies in patient plasma
ot/- and Watsu fc, after adsorption it is 20 cmot/
It only went down to ml.

Comparative Example 5 CpG350 (average pore diameter 345'') as porous glass
Activation with silane and fixation of tryptophan were performed in the same manner as in Example 4, except that A) was used without dissolving with NaOH. The amount of immobilized tryptophan is 32
μmo, j/are.

This adsorption process has a pore diameter of 60 to 15,000X (0.2d to 5
Total pore volume M (1,020C/?) in the pore size range of 0d)
) are distributed, but when the pore size iD is in the pore size range of 60 to 15000X, the maximum value in the pore volume range of 0.8D to 1.2D is the total pore volume - In other words, the pore size distribution was very sharp.

When an adsorption experiment was conducted in the same manner as in Example 4, the anti-acetylcholine receptor antibody in the patient's plasma was 25 cmot.
/ml, whereas after adsorption it was 15 p mol
/ml.

[Brief explanation of the drawing]

The drawing is a cross-sectional view showing an example of an adsorption device using the entire body fluid purifying adsorbent of the present invention. Agent Takeshi Shimizu

Claims (1)

[Claims] In a porous adsorbent having a surface capable of binding to an adsorbed substance, when the diameter of the adsorbed substance is 1 d, 70 or more of the total pore volume of the adsorbent is distributed in a pore size range of 0.2 d to 50 d. and, when the pore diameter is 1 kD, the pore volume in the range of 0.8D to 1.2D is less than 80% of the total pore volume at any pore diameter.