JPH02149341A - Adsorbent for serum amyloid p-protein - Google Patents

Abstract

PURPOSE:To obtain an economical adsorbent for serum amyloids P-protein which selectively remove the serum amyloids P by fixing an anionic functional group-containing compound in a water insoluble carrier. CONSTITUTION:In an adsorbent to remove serum amyloids P-protein contained in blood, compounds of agarose, dextran, etc., having hydroxy groups and removal limiting molecular weight >=230000 are used as a water insoluble carrier. Then, polyanionic compounds having anionic functional groups such as sulfate ester groups, sulfonyl, etc., are fixed in the carrier. Adsorbents prepared by this way remove serum amyloids P-protein selectively and are economical.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to serum amyloid P (Se
The present invention relates to an adsorbent for SAP protein for removing rum Amyloid Ps (hereinafter referred to as SAP) protein.

[Problems to be solved by conventional technology and invention]

Amyloidosis is a phenomenon in which β-fibrillar proteins called amyloid substances are deposited in blood vessels, organs, and other tissues, leading to organ failure such as the heart and kidneys, cardiac conduction disorders, progressive dementia, cerebrovascular disorders, neurological disorders, etc. It is a disease that causes serious disability.

It is known that amyloidosis has disease types such as primary, persistent, familial, and senile, and the protein organization of amyloidosis deposits differs depending on the disease type. Also,
It is becoming clear that precursor substances corresponding to the amyloid substances deposited in each disease type exist in patient blood.

In the 1960s, it was discovered that there are two different types of amyloid substances: one is fibrillar (amyloid f'brils), and the other is pentagonal rod-shaped (P-coIlponentx A).
An electron microscopic search revealed that P) exists. In other words, a substance called amyloid P protein (AP) is found bound to β-fibrillar proteins in all types of amyloidosis, except for intracerebral plaques in Alzheimer's disease and senile dementia. ing. A
Regarding the structure of P, it has been investigated that a pentagonal unit is formed from five subunits with a molecular weight of 23,000 to 25,000, and that a pair of units are arranged in parallel to form a 100 total.

It has been confirmed by various methods that this AP is identical to SAP, which is a normal human plasma protein. Therefore, AP is thought to be a tissue-deposited version of circulating SAP, ie, SAP is a precursor of AP. Although the functions in vivo and the relationship between these molecules and the deposition of other amyloid substances are not well understood, there are reports that AP is an integral part of amyloid fibrils, and that SAP deposition may be associated with amyloid- It seems to have a major significance in the onset of the disease.

As mentioned above, amyloidosis is a serious disease with a high mortality rate, so treatments for it have been actively researched, but so far no effective treatment, especially drug therapy, has been found. .

On the other hand, attempts have been made to treat amyloidosis using blood purification methods using extracorporeal circulation, which have become popular in recent years, especially plasmapheresis. It has been reported that replacing the tube reduces symptoms and halts the progression of lesions.

Blood purification through extracorporeal circulation, especially plasmapheresis, is currently the most effective treatment, but it requires the use of large amounts of expensive and valuable normal plasma or plasma preparations, and the use of precursors other than those contained in the patient's plasma. Since useful components are also discarded at the same time, there is a strong desire to develop a method for more selectively removing precursors such as SAP protein.

The object of the present invention is to solve the problem of slanting and provide an inexpensive adsorbent for SAP protein that selectively removes SAP m white.

[Means to solve the problem]

The present invention relates to an adsorbent for SAP protein in which a compound having an anionic functional group is immobilized on a water-insoluble carrier.

〔Example〕

In this specification, body fluids include blood, plasma, serum, ascites, lymph, intraarticular fluid, and fractionated components obtained therefrom.
and other biologically derived humoral components.

The water-insoluble carrier used in the present invention is preferably porous, particularly one having continuous pores of large diameter. In other words, the subunits of SAP protein form a decamer and the molecular weight reaches 280,000 to 250,000, so in order to efficiently adsorb it, the SAP protein must easily penetrate into the porous carrier. It is necessary.

Exclusion limit molecular weight is often used as a measure of pore size. What is the exclusion limit molecular weight?
As stated in the following, it refers to the molecular weight of the smallest molecular weight of the molecules that cannot enter (excluded) the pores in gel permeation chromatography.

Therefore, the water-insoluble porous carrier used in the present invention preferably has an exclusion limit molecular weight of 230,000 or more (a value obtained using a globular protein, the same applies hereinafter).

On the other hand, those with an exclusion limit molecular weight of more than 100 million tend to be unsuitable for practical use because the mechanical strength of the adsorbent is weakened or the solid content of the adsorbent is too small to provide sufficient adsorption capacity. be. Therefore, the exclusion limit molecular weight is preferably 100 million or less, more preferably 50 million or less.

Next, regarding the porous structure of the water-insoluble carrier, total porosity is preferable to surface porosity, and the pore volume is preferably 20% or more from the viewpoint of high adsorption capacity. The shape of the water-insoluble carrier can be selected from any shape such as granules, spheres, fibers, membranes, and hollow fibers. When using a particulate water-insoluble carrier, the pressure drop is large if the particle size is less than 1, and the adsorption capacity is small if it exceeds 5000.17111, so the particle size should be 1-5000.
It is preferable that it is below am.

The water-insoluble carrier used in the present invention may be either organic or inorganic, but it is preferably one that has little adsorption (so-called non-specific adsorption) of body fluid components other than the target SAP protein. A water-insoluble carrier is preferably hydrophilic rather than hydrophobic, since non-specific adsorption is less likely to occur if the carrier is hydrophilic, and a water-insoluble carrier made of a compound having a hydroxyl group in the molecule is more preferred.

Typical examples of water-insoluble carriers used in the present invention include soft porous carriers such as agarose, dextran, and polyacrylamide, inorganic porous carriers such as porous glass and porous silica gel, polymethyl methacrylate, polyvinyl alcohol, and styrene. Examples include, but are not limited to, porous polymer hard gels made from synthetic polymers such as divinylbenzene copolymers and/or natural polymers such as cellulose.

When using the adsorbent of the present invention for extracorporeal circulation treatment, blood,
Because it is necessary to flow an anti-viscous fluid such as plasma at high speed,
Preferably, a rigid water-insoluble carrier is used that has sufficient mechanical strength not to cause compaction. In other words, a hard carrier is a water-insoluble carrier that is uniformly packed into a cylindrical column and has a linear relationship between pressure drop and flow rate up to at least 0.3 kg/C when an aqueous fluid is passed through it, as shown in the reference example below. Refers to things that are in a relationship.

Any anionic functional group used in the present invention may be used as long as it is negatively charged at around neutral pH. Typical examples of these include, but are not limited to, carboxyl groups, sulfonic acid groups, sulfone groups, sulfuric ester groups, silanol groups, phosphoric ester groups, and phenolic hydroxyl groups.

Among these, carboxyl groups, sulfonic acid groups, sulfate ester groups, and phosphate ester groups are preferred because they have a strong affinity for SAP protein.

As a compound having an anionic functional group, 1
It may be a monoanionic compound having one anionic functional group or a polyanionic compound having multiple anionic functional groups. Polyanionic compounds are preferred because they have a high affinity for SAP protein and can easily introduce many anionic functional groups into a unit amount of carrier.

Among these, polyanionic compounds having a molecular weight of 1000 or more are preferred in terms of affinity and the amount of anionic functional group introduced. The polyanion compound may have one or two types of anionic functional groups.

Representative examples of polyanionic compounds used in the present invention include:
Synthetic polyanionic compounds such as polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, styrene-maleic acid copolymer, and anionic functional group-containing polysaccharides such as heparin, dextran sulfate, chondroitin, chondroitin sulfate, phosphomannan, chitin, and chitosan, but are not limited thereto.

The number of compounds having anionic functional groups immobilized on the adsorbent of the present invention may be one type, or two or more types.

The adsorbent of the present invention is one in which a compound having an anionic functional group is immobilized on a water-insoluble carrier. There are various methods of introducing an anionic functional group into an adsorbent to obtain a fixed state of a compound having such an anionic functional group. (1) A method of forming an adsorbent by polymerization using an anionic functional group or a compound containing a functional group that can be easily converted into an anionic functional group as a monomer or a crosslinking agent; (2) A method containing an anionic functional group. (3) A method of immobilizing a compound having an anionic functional group on a water-insoluble carrier by directly reacting the compound forming the anionic functional group with the water-insoluble carrier. can give.

Of course, an anionic functional group-containing compound such as glass, silica, alumina, etc. that originally contains an anionic functional group may be used as the adsorbent.

Typical examples of monomers or crosslinking agents containing anionic functional groups or functional groups that can be easily converted into anionic functional groups used in method (1) include acrylic acid and its esters, methacrylic acid and its esters, and styrene. Examples include, but are not limited to, sulfonic acids.

Method (2), that is, a method for immobilizing a compound containing an anionic functional group on a water-insoluble carrier, includes physical adsorption, ionic bonding, and covalent bonding. However, in order to use the adsorbent for therapeutic purposes, it is important that the anionic functional group-containing compound does not come off during sterilization or treatment, so a covalent bonding method that allows for strong immobilization is preferred.

When an anionic functional group-containing compound is immobilized by a covalent bond, it is preferable that the anionic functional group-containing compound has a functional group other than the anionic functional group that can be used for immobilization.

Typical examples of functional groups that can be used for immobilization include amino groups,
Examples include, but are not limited to, amide groups, carboxyl groups, acid anhydride groups, succinylimide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, and silanol groups.

There are many anionic functional group-containing compounds having these functional groups, examples of which include taurine, sulfanilic acid, glycine, phosphorylethanolamine, and tyrosine.

Among compounds containing anionic functional groups, typical examples of compounds containing sulfate groups include sulfate esters of hydroxyl group-containing compounds such as alcohols, sugars, and glycols, among which polyhydric alcohols Partially sulfated esters of saccharides, especially sulfated saccharides, contain both sulfate groups and functional groups necessary for immobilization, and have high biocompatibility and activity.
Furthermore, sulfated polysaccharides are particularly preferred because they can be easily immobilized on water-insoluble carriers.

Next, method (3) is used, that is, by reacting a compound that forms an anionic functional group with a water-insoluble carrier, the compound having an anionic functional group is immobilized on the water-insoluble carrier, and the anionic functional group is introduced. A typical example of the method is a reaction in which a sulfate ester group is introduced into a hydroxyl group-containing carrier. In this case, a sulfate ester group can be directly introduced by reacting a hydroxyl group-containing water-insoluble carrier with a reagent such as chlorosulfonic acid or concentrated sulfuric acid.

There are various methods for removing SAP protein from body fluids using the adsorbent of the present invention, and any method may be used. SA in which a compound having an anionic functional group is fixed to a water-insoluble porous material that can pass through
S consisting of a filter through which the P protein adsorbent cannot pass, and the SAP protein adsorbent filled in the container.
A method of passing body fluid through an AP protein removal device is simple and preferred.

Reference Example: A glass column (inner diameter: 9 ml, column length: 150 m) equipped with a filter with a pore diameter of 15 at both ends, agarose gel (Biogel A5m: trade name, manufactured by Bio-Rad, particle size: 50 to 100 mesh), a gel made of a synthetic polymer, Toyopearl OWE5 (trade name, manufactured by Tosoh ■, particle size 50-100 l!m') and porous cellulose gel, Cellulofine GC-700 (trade name, manufactured by Chisso ■, particle size 45-100JI) were each uniformly applied. The column was filled, water was passed through the column using a peristaltic pump, and the relationship between flow rate and pressure loss ΔP was determined. The results are shown in FIG. As is clear from the figure, agarose gel, which is a soft gel, undergoes consolidation when the flow rate exceeds a certain level, and the flow rate does not increase even if the pressure is increased, whereas hard gels such as Toyopearl and Cellulofine undergo compaction when the flow rate exceeds a certain level. The flow rate increases approximately in proportion to the increase.

Production Example 1 CK Gel A3 (trade name,
Made by Chisso ■, exclusion limit molecular weight of globular protein 50 million,
Particle size 83-125 lJm) 100 ml of water
m1゜2N sodium hydroxide aqueous solution 51ml. 18 ml of epichlorohydrin was added, and the mixture was stirred at 40°C for 2 hours.

After the reaction, the gel was filtered and washed with water to form epoxidized CK gel A3.
(hereinafter referred to as epoxidized gel) was obtained.

Example 1 To 5 ml of the epoxidized gel obtained in Production Example 1, a solution prepared by dissolving 0.17 g of sulfanilic acid in 10 ml of water and adjusting the pH to 9.9 was added, and the mixture was shaken at room temperature for 24 hours, resulting in a concentration of 0.5%
A monoethanolamine aqueous solution was added and shaken to seal unreacted epoxy groups to obtain a cellulose gel in which sulfanilic acid was immobilized.

Example 2 5 ml of the epoxidized gel obtained in Production Example 1 was added with a molecular weight of about 5.
000, add 4 g of dextran sodium sulfate containing 18% sulfur and 5 ml of water 1) Adjust to H9 and make 45
Shake at °C for 18 hours. After that, the gel was filtered and 2M
Wash with saline solution, 0.5M saline solution, and water in this order, add 0.5% monoethanolamine aqueous solution, and shake at room temperature to seal unreacted epoxy groups and fix dextran sodium sulfate. A cellulose gel was obtained.

Production Example 2 A porous cellulose carrier of the same type as that used in Production Example 1 (
CK Gel A3) 40 ml was suspended in heptane to make the total volume 70 ml. To this was added 10 ml of 20% NaOH and 40 drops of nonionic surfactant Tween 20 (trade name, manufactured by Bio-Rad), and the mixture was shaken at 40'C for 30 minutes. Next, add 10ml of epichlorohydrin at 40°C.
The mixture was shaken for 6 hours. After the reaction was completed, the gel was filtered and washed with ethanol and water in that order to obtain an epoxidized gel.

Example 3 Add polyacrylic acid (molecular weight approximately 1000) having an amino group at one end to 10 ml of the epoxidized gel obtained in Production Example 2.
Dissolve 1 g in 5 ml of water and add 2M Na
1 ml of OH was added and the mixture was left at room temperature for 48 hours.

After the reaction was completed, the gel was filtered and washed with water to obtain a cellulose gel on which polyacrylic acid was fixed. Polyacrylic acid with an amino group introduced at one end was obtained from a low polymerization reaction of acrylic acid using 2-aminoethanethiol as a chain transfer agent and azobisisobutyronitrile (AIBN) as an initiator (Nippon Kagaku Co., Ltd. ``Synthesis of 2-hydroxyethyl-methacrylate-styrenic ABA type block copolymer and its structure and wettability'', Mitsuo Okano et al., Journal of the Society of Japan, 1977, pp. 88-92).

Example 4 Water was added to 5 ml of the epoxidized gel obtained in Production Example 1 to make the total volume 9 ml. After dissolving 0.09 g of L-tyrosine in this, 2N NaOH was added to adjust the pH to 9, and the pH was adjusted to 2 at room temperature.
I left it for days. After the reaction was completed, the gel was filtered and washed with water to obtain a cellulose gel on which tyrosine was fixed.

Example 5 Adsorbent and CK gel AS O obtained in Examples 1 to 4
, was placed in a polypropylene test tube, 0.5 ml of human serum containing SAP protein was added thereto, and the mixture was shaken at 37°C for 2 hours. After shaking, the SAP protein concentration of the supernatant was measured by solid-phase enzyme-linked immunosorbent assay (ELISA).

That is, first add diluted anti-SAP protein antibody (
(manufactured by Hoechst) was added dropwise, and the antibody was fixed on the plate by leaving it at 4° C. overnight.

Next, the diluted sample was dropped onto the plate on which the anti-SAP protein antibody was immobilized, and the antigen-antibody reaction was carried out at room temperature for 1 hour. After washing, the peroxidase-labeled anti-SAP protein antibody was dropped and the antigen-antibody reaction was carried out in the same way at room temperature. After washing for 1 hour,
Perform an enzymatic color reaction and measure the degree of color development using C8-930.
(Product name, manufactured by Shimadzu Corporation) (absorbance: 49)
2 nm). Table 1 shows the compounds having anionic functional groups fixed on each adsorbent and the SAP of each adsorbent.
Shows the adsorption rate. The SAP protein adsorption rate is expressed as the rate of decrease in the supernatant SAP protein concentration of each adsorbent relative to the supernatant SAP protein concentration of CK gel A3. From the results in Table 1, it can be seen that the adsorbent of the present invention adsorbs SAP protein very well.

Table 1 [Effects of the Invention] The adsorbent of the present invention is inexpensive and can absorb SA contained in body fluids.
This has the effect of selectively removing P protein.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of investigating the relationship between flow velocity and pressure loss using three types of gels. Patent applicant Kanebuchi Chemical Industry Co., Ltd.

Claims (1)

[Scope of Claims] 1. An adsorbent for serum amyloid P protein, comprising a compound having an anionic functional group immobilized on a water-insoluble carrier. 2. The adsorbent for serum amyloid P protein according to claim 1, wherein the water-insoluble carrier is a porous material. 3. The adsorbent for serum amyloid P protein according to claim 1 or 2, wherein the anionic functional group consists of at least one type selected from the group consisting of a sulfate ester group, a sulfonic acid group, a carboxyl group, and a phosphate ester group. . 4. The adsorbent for serum amyloid P protein according to claim 1 or 2, wherein the compound having an anionic functional group is a polyanionic compound having a plurality of anionic functional groups in one molecule. 5. The adsorbent for serum amyloid P protein according to claim 1 or 2, wherein the water-insoluble carrier comprises a compound having a hydroxyl group.