JPH01171638A - Adsorbent for serum amyloid a protein - Google Patents

Abstract

PURPOSE:To selectively adsorb serum amyloid A protein without decreasing high density lipoprotein, by forming adsorbent for serum amyloid A protein by contg. polyanion compd. at least on a part of the surface of water-insoluble carrier. CONSTITUTION:The polyanion compd. is contained at least on a part of the surface of the water-insoluble carrier such as inorg. carrier such as glass beads, silica gel, synthetic polymer such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, org. carrier consisting of polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, to form the absorbent for serum amyloid A protein. The representative example of used polyanion compd. is synthetic polyanion compd. etc., such as polyacrylic acid, polyvinyl sulfonic acid. As the means for introducing the polyanion compd., a fixing method forming a strong covalent bond is suitable.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to serum amyloid A (Se
rum AmyloId A, , hereinafter referred to as SAA)
The present invention relates to an adsorbent for SAA proteins for removing proteins.

[Prior art and problems to be solved by the 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 in the heart, kidneys, etc., and cardiac stimulation. It is a disease that causes serious disorders such as conduction disorders, progressive dementia, cerebrovascular disorders, and neurological disorders.

It is known that amyloidosis has disease types such as primary, secondary, familial, and senile. Secondary amyloidosis occurs secondary to diseases such as chronic rheumatoid arthritis, juvenile rheumatoid arthritis, pulmonary suppuration, and pulmonary tuberculosis, and frequently occurs in the Y, thyroid, pancreas, liver, and tablets.

The protein organization of amyloid-cis deposits differs depending on the disease type. In secondary amyloidosis, the amyloidosis deposits are amyloid A (Amyloid A).
It is made up of a protein called AA protein, which has a molecular weight of 11,500 and consists of 76 amino acids.

Furthermore, it is becoming clear that precursor substances corresponding to amyloid-cis deposits deposited in each disease type exist in patient blood. In secondary amyloidosis, high-density lipoprotein (IIIgh Der+5ity
SAA protein, which is one of the apolipoproteins of lipoproteins (hereinafter referred to as HDL5), is said to be a precursor of AA protein.
approximately 1.2000, produced in liver cells and iID in the blood
It is thought that it circulates in the body as an apolipoprotein of I, and then is separated from L and hydrolyzed, becoming AA protein and deposited in tissues. Also SAA ffl white is It
It is believed that the medium DLJ exists as a dense apolipoprotein of IIDLJ with a molecular weight of 175/105.

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. do not have.

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, particularly plasma exchange methods, in which patient plasma containing large amounts of the aforementioned precursors is exchanged with normal plasma. It has been reported that symptoms are alleviated and the progression of lesions is halted.

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 it requires the use of precursors other than those contained in the patient's plasma. However, there is a strong demand for the development of a method for more selectively removing the precursors, as the useful components of the precursors are also discarded at the same time.

The present invention solves the above problems and provides l! The object of the present invention is to provide an inexpensive adsorbent for SAA protein that can selectively remove SAA protein without significantly reducing DL.

Means for Solving Problem L] The present invention relates to an adsorbent for SAA protein having a polyanionic compound on at least a portion of the surface of a water-insoluble carrier.

[Example] The polyanion compound used in the present invention is not particularly limited, and may be any compound containing a functional group that is negatively charged when the pH is near neutral.

Representative examples of polyanionic compounds used in the present invention include:
For example, polyacrylic acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid, polyglutamic acid, polyaspartic acid,
Examples include synthetic polyanionic compounds such as polymethacrylic acid, polyphosphoric acid, and styrene-maleic acid copolymers, as well as polysaccharides containing anionic functional groups such as heparin, dextran sulfate, chondroitin, chondroitin sulfate, chitin, and chitosan. , but not limited to these.

Among these, it is preferable to use a polyanionic compound containing a sulfate ester group.

Various methods can be used to introduce the polyanionic compound to at least a portion of the surface of the water-insoluble carrier, but an immobilization method that forms a strong covalent bond is preferred.

Among the polyanion compounds, the compound containing a sulfate ester group is preferably a compound having a functional group that can be used for immobilization on a water-insoluble carrier in addition to the sulfate ester group. Among them, partially sulfated esters of polyhydric alcohols, especially sulfated saccharides (sulfated polysaccharides), contain both sulfate groups and functional groups necessary for fixation, and are highly biocompatible and active. Furthermore, it is preferable because it can be easily immobilized on a water-insoluble carrier. Other polyanionic compounds preferably have a functional group that can be used for fixation in addition to the anionic functional group.

The water-insoluble carrier used in the present invention may be an inorganic carrier, an organic carrier consisting of a synthetic polymer or polysaccharide, or a composite carrier consisting of an organic carrier and an inorganic carrier, but the presence of SAA protein present in blood From an environmental point of view, a hydrophilic water-insoluble carrier is preferred, and a carrier with less adsorption of substances other than the target substance, so-called non-specific adsorption, is more preferred. Furthermore, it is advantageous if a functional group that can be used for the immobilization reaction or a functional group that can be easily activated exists on the surface of the carrier. Representative examples of these functional groups include amino group, carboxyl group, hydroxyl group, thiol group, acid anhydride group, succinylimide group, salt tQ L%, aldehyde group,
Examples include amide group, eboxy group, and silanol group.

Preferably used in the present invention 1. Representative examples of water-insoluble carriers and (-
Inorganic carriers such as glass beads and silica gel, synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, and cross-linked polyamide, and organic carriers made of polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran, etc. Furthermore, composite carriers of organic carriers and inorganic carriers whose surfaces are coated with synthetic polymeric compounds or polysaccharides, and composite carriers of organic carriers and organic carriers whose surfaces are coated with polysaccharides made of synthetic polymeric compounds. However, the present invention is not limited to these. Among these carriers, a water-insoluble carrier composed of a hydroxyl group-containing compound is particularly preferred because of its good biocompatibility and low nonspecific adsorption.

Is the shape of the adsorbent of the present invention granular, fibrous, film-like, or hollow? The adsorbent may have any shape, such as a fiber-like shape, and the fine structure of the adsorbent may be porous or non-porous6, but in order to obtain a high adsorption capacity per unit volume, it is necessary to Preferably, it is large, ie porous.

The adsorbent of the present invention can be used by subjecting blood, serum, plasma, diluted liquids thereof, or these liquids to pretreatment such as blood cell removal and serum protein removal. It can be used to remove SAA proteins from solutions containing SAA proteins, such as
It can also be used as an adsorbent for extracorporeal circulation treatment of patients with secondary amyloidosis. For example, when used as an adsorbent for in vitro seedling treatment, it must have sufficient mechanical strength to prevent compaction of the adsorbent, and must also have porous properties to obtain a high adsorption amount per unit volume. Preferably, it is an adsorbent having (7. Preferably, the gel is a rigid gel and is porous, especially completely porous, and the pore size is large enough to allow the SAA protein with a molecular weight of 12,000 to penetrate into the gel. It is preferable.

As shown in the reference example below, the hard gel referred to here is made by uniformly filling a cylindrical column with gel and flowing an aqueous fluid through it.

The relationship between pressure loss and flow rate is linear up to 0.3 kg/cm'. Furthermore, porous means that the pore volume is 20% or more and the specific surface area is 3 ni'/g or less, and the pore diameter is large enough for SAA protein to penetrate into the gel. , means a size with an exclusion limit molecular weight of 12,000 or more (a value obtained using a globular protein), which is often used as a guideline for the pore diameter of a gel. Furthermore, it is preferable that the exclusion limit molecular weight is 300,000 or more, since this facilitates the diffusion of SAA protein into the gel.

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.

The exclusion limit molecular weight is a molecular weight that cannot penetrate into the pores in gel permeation chromatography, as described in "Experimental High-Performance Liquid Lichromatography" (co-authored by Fuyuki Hatano and Toshihiko Hana, published by Mari Kagaku Dojin). In other words, it refers to the molecular weight of the one having the smallest molecular weight among the excluded molecules.

Next, the adsorbent of the present invention and its manufacturing method will be explained in more detail based on Examples, but the present invention is not limited to these.

Reference example A glass cylindrical column (inner diameter 9+am, column length 1501!1) equipped with a filter with a pore diameter of 151M1 at both ends
111) on agarose gel (Bolorad
o) Biogel A301. Particle size 50-100
Polymer hard gel (Toyo Pearl HW65 manufactured by Toyo Soda Kogyo ■, particle size 50-100ρ, and Cellulofine GC-700 manufactured by Chisso ■, particle size 45-105
(Additional) was filled uniformly in each case, water was flowed through the tubes using a veristatic pump, and the relationship between the flow rate and the pressure loss ΔP was determined. The results are shown in FIG.

From the results shown in FIG. 1, it can be seen that the flow rate of the polymer hard gel increases almost in proportion to the increase in pressure, whereas the agarose gel causes compaction and the flow rate does not increase even if the pressure is increased.

Example 1 CK gel A-3 (trade name, porous cellulose gel)
Manufactured by Chisso ■, globular protein exclusion limit molecule Q50 million,
Particle size 63~12! ga) 100ml of water per 100m1
ml, 53 ml of 2N aqueous sodium hydroxide solution and 18 ml of epichlorohydrin were added, and the mixture was stirred at 40° C. for 2 hours.

After the reaction, the gel was filtered and washed with water to obtain epoxidized CK gel A-3.
I got it.

Add 46.5 g of dextran sodium sulfate and a small amount of water to 100 ml of the obtained epoxidized CK gel A-3 to completely dissolve it, and then add more water to bring the total volume to 177.
Adjusted to ml. I)H with 2N aqueous sodium hydroxide solution
After adjusting the yS to 9.2, it was left standing at 45°C for 16 hours. After the reaction, the gel was filtered and washed with water to remove dextran sulfate immobilized C.
I obtained K gel A-3.

0.4 ml of the resulting dextran sulfate-immobilized gel was placed in a test tube, 2.4 ml of human serum containing SAA protein was added, and the mixture was shaken at 37°C for 2 hours. After shaking, the concentration of SAA protein in the supernatant was measured.

SAA and protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) as described below. That is, the plate was first diluted with anti-SAA antibody (Hoechst).
Antibodies were immobilized on the plate by adding dropwise solution (manufactured by St. Co., Ltd.) and allowing the plate to stand at 4°C for 12 hours. Next, the diluted specimen and standard serum were added dropwise and allowed to stand at room temperature for 3 hours to perform an antigen-antibody reaction.
Antibody A was added dropwise and the antigen-antibody reaction was similarly carried out at room temperature for 3 hours. After washing, an enzymatic color reaction is performed, and the degree of color development is measured using C5-930 (trade name, manufactured by Shimabara Seisakusho) at wavelength 4.
Measured at 92 nm. By comparing the degree of color development for the sample and the degree of color development for the standard serum, the SAA protein concentration in the sample was determined, assuming that the concentration of SAA protein in the standard serum was 1. Also, to see the selectivity, HD
The concentration of L-cholesterol was also measured.

The results are shown in Table 1.

Example 2 CK gel A-22 instead of CK gel A-3100ml
(Product name, manufactured by Chisso Oki, globular protein exclusion limit molecule m
30 million, particle size 53-12FBam) loOm
Dextran sulfate-immobilized CK gel A-22 was obtained in the same manner as in Example 1 except that 1 was used.

The obtained dextran sulfate-immobilized gel was treated in the same manner as in Example 1, and SAA protein and HDL-
The concentration of cholesterol was measured.

The results are shown in Table 1.

Example 3 Epoxidized CK gel A-3100m obtained in Example 1
15.6 of polyacrylic acid having an amino group at one end
g, and then water was added to bring the total volume to 156 ml. After shaking well, the mixture was allowed to stand at 50°C for 10 hours. After the reaction, the gel was filtered and washed with water to obtain polyacrylic acid-immobilized GK gel A-3.

The resulting polyacrylic acid immobilized gel was treated in the same manner as in Example 1, and the concentrations of SAA protein and HDL-cholesterol were measured.

The results are shown in Table 1.

Comparative Example 1 Dextran sulfate-immobilized CK gel A obtained in Example 1
The SAA protein and HD L-cholesterol concentrations of the supernatant obtained in exactly the same manner as in Example 1 except that 0.24 ml of physiological saline was used instead of -30.4 ml were measured.

The results are shown in Table 1.

[Left below] Table 1 From the results in Table 1, when the adsorbent of the present invention is used, SAA f
It can be seen that fi white is adsorbed, but IIDL is hardly adsorbed.

[Effects of the Invention] The adsorbent of the present invention is inexpensive and has the effect of being able to selectively remove SAA 1 contained in body fluids without significantly reducing IIDI.

[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. Figure 1 Pressure loss Pc kg/Cm2)

Claims (1)

[Claims] 1. An adsorbent for serum amyloid A protein having a polyanionic compound on at least a portion of the surface of a water-insoluble carrier. 2. The adsorbent according to claim 1, wherein the water-insoluble carrier is composed of a hydroxyl group-containing compound. 3. The adsorbent according to claim 1, wherein the polyanion compound contains a sulfate ester group. 4. The adsorbent according to claim 1, wherein the polyanion compound is fixed to a water-insoluble carrier by a covalent bond. 5. The adsorbent according to claim 3, wherein the sulfate group-containing compound is a sulfated polysaccharide. 6 The water-insoluble carrier is
The adsorbent according to claim 1, having an exclusion limit molecular weight of 300,000 to 50,000,000.