JPS6282973A - Material and apparatus for adsorbing beta-lipoprotein - 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] ■. BACKGROUND OF THE INVENTION [Technical Field] The present invention relates to a novel β-riboprotein adsorbent and a β-riboprotein adsorption device using the adsorbent.

β-riboprotein accounts for approximately 5-7% of human plasma proteins;
It is known that the amount of cholesterin in the blood increases as we age, leading to arteriosclerosis. Also,
Familial hypercholesterolemia, an inherited disease, exhibits β-riboprotein levels several times the normal value and is susceptible to coronary artery occlusion.

Therefore, by specifically adsorbing and removing β-riboproteins from body fluids such as blood and plasma, we prevent the progression of the above-mentioned diseases.
It is expected to alleviate symptoms and even speed up treatment. The adsorbent and adsorption device of the present invention can be effectively used for such therapy.

[Prior art and its problems] Conventionally, there have been many reports of adsorbents that can be used for such purposes, in which polysaccharides (polyanions) such as heparin, dextran sulfate, chondroitin sulfate, hyaluronic acid, and alginic acid are immobilized on carriers. has been done. For example, a low-density riboprotein (LDL) adsorbent in which the above-mentioned polyanion is immobilized on a carrier made of an inorganic porous material, cellulose, or polyvinyl alcohol-vinyl acetate copolymer is disclosed in JP-A-59
-156431 publication, 59-193135 publication,
It is disclosed in JP 59-196738, JP 59-197255, and JP 59-20 (3045j). However, none of these conventional adsorbents have sufficient adsorption capacity or adsorption selectivity for C, and The emergence of an excellent β-riboprotein adsorbent has been desired.

■8 Purpose of the Invention In view of the above-mentioned problems of conventional adsorbents, an object of the present invention is to provide an adsorbent and an adsorption device that can adsorb β-riboprotein with high activity and more specifically. .

A further object of the present invention is to provide an adsorbent and an adsorption device Y that maintain stable adsorption capacity, are safe, can be easily sterilized, and are suitable for body fluid purification or regeneration.

The adsorbent and suction unit U of the present invention have the following configuration in order to achieve the object described in F.

(1) A β-riboprotein adsorbent comprising a compound having a negative charge substituent immobilized on an insoluble carrier.

(2) A compound with a negatively charged exchange group is a sulfone [
2. The adsorbent for β-riboprotein according to item 1, which is i and/or a carboxyl compound.

(3) The β-riboprotein adsorbent according to item 1 or 2, wherein the compound having a negatively charged substituent has an aromatic ring.

(4) Compounds with negatively charged substituents are sulfone M
4. The β-riboprotein adsorbent according to item 3, which is a benzene-based aromatic compound 2 having a group and a carboxyl group.

(5) The β-riboprotein adsorbent according to item 4, wherein the compound having a negatively charged @ substituent is 4-sulfosalicylic acid.

(6) β-, which is formed by immobilizing a compound having a negatively charged substituent on an insoluble carrier in a volume having a fluid inlet and outlet.
A β-riboprotein adsorbent characterized by containing a riboprotein adsorbent? 'I device.

(7) The β-riboprotein absorption device 7 according to item 6, wherein the container is filled with ice cubes and sterilized in an autoclave.
1.

■． Detailed Description of the Invention The target adsorbed substance of the present invention is β-riboprotein, and more specifically, α1, α2 and β-globulin fractions observed when serum lipids are separated by electrophoresis. Low-density riboprotein (LDL) and very low-density riboprotein (V) contained in the β-globulin fraction
LDL). LDL- and VLDL contain large amounts of cholesterol and triglycerides, which cause arteriosclerosis.

The compound having a substituent with a negative charge used as an adsorbent (ligand) in the present invention means that when immobilized on an insoluble carrier, the compound has a physiological pl+ region (neutral (e.g. blood, body fluid)).
] refers to a compound whose surface potency p exhibits a negative value. A compound with a negatively charged exchange group (hereinafter referred to as a negatively charged compound) is a low-molecular organic compound that has a sulfonic acid group and/or a carboxyl group in its molecule, especially a low-molecular compound that has an aromatic ring. is preferred. Low-molecular-weight compounds are preferred because they are particularly easy to sterilize using O-crepe after being assembled into a device. Examples of low-molecular-weight compounds containing aromatic rings include benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene; oxygen-containing aromatic rings such as dibenzofuran, fromene, and benzofuran;
Sulfur-containing aromatic rings such as thianthrene, phenanthrene,
Examples include nitrogen-containing 6 C1 rings such as quinoline and acridine, and nitrogen-containing 5-membered rings such as indole and carbazole. Particularly preferred compounds include sulfone 1 in the benzene aromatic ring.
1 and a compound into which a carboxyl group has been introduced. Examples include benzoic acid, toluic acid, salicylic acid, phthalic acid, benzenesulfonic acid, toluenesulfonic acid, and benzenedisulfonic acid, and particularly preferred is sulfonesalicylic acid having both a sulfone MP, S and a carboxyl group.

In the present invention, the insoluble carrier can be either a woody carrier or a hydrophobic carrier.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but from the viewpoint of permeability of blood and plasma, ease of handling when preparing the adsorbent, etc. , particulate carriers are particularly preferably used.

The particulate carrier preferably has an average particle diameter of 0.05 to 5. Average particle size is JIS-2-88
The intermediate value between the upper limit particle size and the lower limit particle size of each grade is taken as the particle size of each grade, and the average particle size is calculated as the weight average.

Further, the particle shape is preferably spherical because it is less likely to damage cells and less likely to cause flaking or chipping, but is not particularly limited.

Particulate carriers that can be used include agarose, dextran, cellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylonitrile, styrene-divinylbenzene copolymer, polybrene, and acrylic esters. There are 10 types including methacrylic acid ester type, glass type, alumina type, titanium type, activated carbon type, and ceramic type, but porous polymer particles, porous glass, silica gel, and porous silica alumina are particularly good. Give results. The polymer composition is
Known polymers that can have a porous structure, such as polyamides, polyesters, polyurethanes, and vinyl compound polymers, can be used. Furthermore, immobilized enzymes and known carriers commonly used in affinity chromatography can be used without any particular limitations.

Within the porous structure, mechanical strength, chemical inertness, surface area,
Particularly preferred is spherical silica gel from the viewpoint of operability.

The porous particles used in the present invention have a negatively charged compound immobilized on their surfaces, and have an average pore diameter of 200 to 15,000 pores, preferably 800 to 30 pores.
00 people.

The porous structure preferably has an average pore size in the range of 200 to 15,000 pores, but if the average pore size is too small, the amount of the substance adsorbed is small; if it is too large, the porous particles It is not practical because the strength and surface area are reduced.

The average pore diameter was measured using a mercury intrusion porosimeter. In this method, mercury is injected into a porous body, and the pore width is determined from the amount of mercury that has entered, and the pore diameter is determined from the pressure required for the intrusion.

Any known method can be used to immobilize a negatively charged compound on the surface of an insoluble carrier, such as coexistence bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the carrier surface. In view of this, it is preferable to use it after it is fixed and insolubilized by covalent bonding.

Various methods are used to covalently bond a negatively charged compound to the surface of an insoluble carrier, depending on the functional groups of both. For example, cyanogen bromide activation method, condensation using carbodiimide reagent/i, etc.: X chemical method, acid azide derivative method, diazo reagent,
Alkylation method, carrier crosslinking method using dialdehyde, his-epoxide, diisocyanate, etc., γ-glycidoxy 10-pyltrimethoxysilane (hereinafter abbreviated as GOPTS) and β-(3,4-1 boxycyclohexyl) ethyltrimethoxysilane A method of activating a silane coupling agent using, for example, is used. Furthermore, if necessary, a molecule of arbitrary length (subaree) may be introduced between the insoluble carrier and the ligand.

A particularly preferred immobilization method is to easily covalently bond to an insoluble support having water fllJ, and to mildly bond with a negatively charged substance having a functional group having active hydrogen such as an amino group, water MW, thiol group, or carboxyl Lt. This method uses the silane coupling agent GOPTS, which forms stable covalent bonds under certain conditions.

The sulfonic acid group and/or carboxyl group of the negatively charged compound preferably used in the present invention causes electrostatic interaction with the positive charge of β-riboprotein, and the aromatic ring has a hydrophobic group of β-riboprotein. They attract each other through hydrophobic interactions.

Furthermore, the hydroxyl groups and terminal hydrogen present in the porous carrier and the coupling agent are attracted to β-riboprotein through hydrogen bonds. In this way, in the adsorption of β-riboprotein and adsorbent,
It can be interpreted that the electrostatic interaction force acts as a main effect, and the hydrophobic interaction force and hydrogen bond force act as side effects, and a favorable result is obtained.

The amount of surface charge on the adsorbent is measured by the streaming potential measurement method, potentiometric titration (DI titration) method, or dye adsorption method described below.

[Streaming potential measurement method]

When a liquid is forced to pass through a capillary filled with adsorbent, a potential difference is created in the direction of the movement of the liquid.

According to the tlclmholtz-3moluchowski equation, the electric potential at the point of shear of the electric double layer, that is, the
The surface potential can be extracted and an approximate value of the surface potential can be obtained.

(Potentiometric titration method) The interfacial electrostatic potential of the adsorbent is determined by performing potentiometric titration using an appropriate alkaline solution.

[Dye adsorption method]

After soaking the adsorbent in a cationic dye solution and rinsing, determine the dye adsorption M and calculate the degree of surface charge (Haroudas
, N.G., ,J.Theor, ,Biol, ,4.
9.417 (1975)).

Although the adsorbent of the present invention can be brought into contact with body fluids and adsorbed and removed unnecessary substances in a batch manner, it is more preferably used in the adsorption device described below.

The β-riboprotein adsorption device of the present invention uses the above-mentioned adsorbent,
It is filled and held in a container equipped with an inlet and outlet for body fluids.

Next, the apparatus of the present invention will be explained with reference to the accompanying drawings.

FIG. 1 is a sectional view showing an example of the device of the present invention.

This apparatus consists of a container 1 having an inlet 4 and an outlet 5 at both ends, an adsorbent 2 housed inside the container, and a filter 3,3' provided at the inlet 4 and outlet 5 to sandwich the adsorbent 2. The filter 3.3' is provided to prevent adsorbent fragments etc. from entering the body fluid and flowing into the body.

The material for the container 1 may be glass, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, etc., but polypropylene, polycarbonate, etc., which can be sterilized by crepe sterilization, are particularly preferred. Furthermore, it is preferable that a filter is provided between the adsorbent layer of the container body and the entrance/exit portion, which has a mesh through which body fluids can pass, but which does not allow the adsorbent to pass through. This material may be physiologically inert and has high strength, but polyester or polyamide is particularly preferably used. The body fluid is introduced through the body fluid inlet 4, treated with the adsorbent 2, and then led out through the body fluid outlet 5. The adsorbent is retained in the container by a filter 3.3'.

For convenience of use, the adsorption device of the present invention is desirably filled with purified water such as distilled water or physiological saline and sterilized in an autoclave to produce a product.

FIG. 2 is a schematic diagram showing an example of blood purification treatment using the adsorption device of the present invention. Blood is in white liquid inlet 6
The blood is then collected and supplied to a plasma separator 8 through a pump 7, where it is separated into white blood and plasma. The separated plasma is supplied to the adsorption container 1 through the pump 7', and after being subjected to adsorption treatment, it is mixed with blood cells in the plasma/blood cell mixing device 9, and is led out from the white liquid outlet 10.

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 plasma components using a centrifuge or a type IIQ or hollow fiber trill device, and then transfer the plasma components to the device. After passing and purifying,
One method is to return the blood to the body together with blood cell components, and the other method is to directly pass the blood taken out from the body through the device and purify it.

The method for passing body fluids may be continuous or intermittent depending on clinical needs or the installation status of the equipment.

Next, the present invention will be explained in more detail with reference to Examples.

Example: To a 2,6W Ha% GOPTS aqueous solution 200ae adjusted to p118 using potassium hydroxide, an average pore size of 2361 particles (particle size range 74-149μ711.Surface area 25m2) was added.
/'J, manufactured by Fuji Davison Chemical) spherical silica gel 70
Mix J and use an aspirator (Tokyo Rikakikai A-28
After degassing with a blood mixer (Kayagaki Medical Science and Engineering T)
The mixture was stirred for 20 minutes using a BM-101 model (manufactured by Co., Ltd.). Next, let it stand for a few minutes, decant the liquid layer, and i
The wet silica gel was transferred to a stainless steel vat, and then heated at 150°C in an A-bun (Model P(S)-212 manufactured by In Leaf Materials) for 1 hour. The activated silica gel 40 (J) thus obtained was dissolved in a mixture (2+3) of distilled water and 9810-0.2M carbonate buffer at 1 W H%.
Bequine, l w/v% gelatin, or 2 w/v
Mix with a solution of V% sulfosalicylic acid and aspirate
I degassed it. Then, after adding 1% pyridine and 1% tri-n-butylamine as a catalyst, the mixture was heated at 80°C.
The mixture was heated with an aqueous solution for 3 hours, and further stirred overnight at room temperature using a Blood Mill 1-Lee to react. Next, after washing with distilled water, 100 ml of a 1M ethanolamine aqueous solution adjusted to pH 8 with acetic acid was added, and the mixture was stirred overnight at room temperature using a blood mixer to block unreacted epoxy groups. Then, the gelatin-bound silica gel was immersed in 100 d of 5M guanidine hydrochloride aqueous solution, and while adjusting ρII to 8 with potassium hydroxide, succinic anhydride 4
．． 95 g was gradually added to succinylate the amino groups of the gelatin. This silica gel bound with succinylated geladin, pectin, and sulfosalicylic acid was washed repeatedly with distilled water, pH 4-0.02M acetate buffer containing 0.5M sodium chloride, and pH 10-0.2M carbonate buffer, and then vacuum-dried at 105°C. It was dried in a container for 3 hours. As a result of the above, an adsorbent (4 (Example)) in which the negatively charged compound sulfosalicylic acid was immobilized, an adsorbent in which pectin was immobilized (Control 1), and an adsorbent in which succinylated gelabun was immobilized (Control 2) were obtained. , the above Examples and Control 1.2 Untreated Tanuki silica gel (Control 3) with nothing fixed on the surface was placed in a glass test tube (Terumo Urubo, 15.5 mm).
Thirteen pieces were weighed out into φx 100 mm and used for the adsorption experiment.

The adsorption experiment was operated as follows. Add 3 d of ril+ 7.2-8 TliLM phosphate buffer (PBS) containing 137 mM sodium chloride and 2.6 mM potassium chloride to the test tube containing the above adsorbent, degas it with an aspirator, and then add 3 d of normal human serum. Add m(l) and heat in a 37°C oven while stirring using a blood mixer.
Incubated for 0 minutes. And capacity fi1.5
It was produced using a disposable syringe in Chill E' of tnQ. Transfer the contents of this test tube to a minicolumn,
Collect the effluent and determine the β-riboprotein concentration and HDL in the effluent.
- Cholesterol 'e4 degree was measured using clinical test kits from β-riboprotein Test Wako and HJ D L-Choleste C1-Route Test Wako. The stone absorption rate was determined by dividing the above measured value by the concentration of the effluent obtained in the same manner without using the adsorbent.

The results are shown in Table 1. From Table 1, it can be seen that the adsorbent that avoids binding sulfosalicylic acid has a higher β
- It is clear that riboproteins are selectively and highly adsorbed and removed.

■2 Effects of the invention The β-riboprotein adsorbent of the present invention can adsorb and remove β-riboprotein, a harmful substance in body fluids, at a high rate and selectively, and is a general material for purifying and regenerating body fluids. It can be used in many cases, and is effective in treating hyperlipidemia, familial hypercholesproleemia, etc.

Furthermore, the β-riboprotein adsorbent of the present invention is not only used for treatment, but can also be effectively used for separating and purifying β-riboprotein and as a material for testing thereof.

Furthermore, the adsorption device of the present invention can easily remove harmful substances from body fluids, and can also perform sterilization operations easily and reliably.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing an example of the suction V of the present invention. FIG. 2 is a schematic diagram showing an example of blood purification treatment using the adsorption device of the present invention.

Claims (7)

[Claims] (1) A β-riboprotein adsorbent comprising a compound having a negatively charged substituent immobilized on an insoluble carrier. (2) The β-riboprotein adsorbent according to claim 1, wherein the compound having a negatively charged substituent is a compound having a sulfonic acid group and/or a carboxyl group. (3) The β-riboprotein adsorbent according to claim 1 or 2, wherein the compound having a negatively charged substituent has an aromatic ring. (4) The β-riboprotein adsorbent according to claim 3, wherein the compound having a negatively charged substituent is a benzene-based aromatic compound having a sulfonic acid group and a carboxyl group. (5) The β-riboprotein adsorbent according to claim 4, wherein the compound having a negatively charged substituent is 4-sulfosalicylic acid. (6) β- formed by immobilizing a compound having a negatively charged substituent on an insoluble carrier in a container having a fluid inlet and outlet
A β-riboprotein adsorption device characterized by containing a riboprotein adsorption material. (7) The β-riboprotein adsorption device according to claim 6, wherein the container is filled with purified water and sterilized in an autoclave.