JPS62244442A - Low specific gravity lipoprotein adsorbing material and its preparation - Google Patents

Abstract

PURPOSE:To suppress the adsorption of a multivalent cation, by using a low specific gravity lipoprotein adsorbing material prepared by mixing a solution of a synthetic chain polymer having a sulfate group adjusted to pH0.5-7 and an activated porous carrier. CONSTITUTION:A low specific gravity lipoprotein adsorbing material is prepared by mixing a solution of a synthetic chain polymer having a sulfate group adjusted to pH0.5-7 and an activated porous carrier being a porous adsorbing material having MW of 5X10<3>-5X10<6>, the synthetic chain polymer part having the sulfate group on the surface thereof and ion exchange capacity of 10-300mueq/ml (wet volume). The aforementioned chain polymer part having the sulfate group is the chain polymer having MW of 5X10<3>-5X10<6> and a large number of sulfate groups in one molecule and, for example, there are polyvinyl sulfate and polystyrene sulfate, etc.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a low-density lipoprotein that selectively adsorbs and removes low-density lipoproteins, which are thought to be closely related to various diseases caused by increased plasma lipids. This invention relates to a specific gravity lipoprotein adsorbent.

As is well known, an increase in blood lipids, particularly low-density lipoproteins, is thought to be closely related to the cause or progression of arteriosclerosis. Myocardial infarction occurs when arteriosclerosis progresses,
The possibility of suffering from serious circulatory system symptoms such as cerebral infarction is extremely high, and the mortality rate is also high. Therefore, by selectively adsorbing and removing low-density lipoproteins from body fluid components such as blood and plasma.

It was expected to prevent the progression of the diseases mentioned above, alleviate symptoms, and even hasten healing.

(Prior art) Existing technologies that can be used for the above purpose include adsorption (Lupie) using an adsorbent in which heharin is immobilized on agarose gel.
n, P-J, et, at, : a new appr
□ach t.

the management of famNiaJ
hypercholesterolemia.

Removal of plasma-chole
sterol based on theprin
cipal of affinity chromat
ography, Lancet.

2:1261 to J264.1976), and chromatography using glass powder or glass beads (Carlson, L. A.: Chromatog.
rapics separation of serum
1ipoprotein on glass powder
er columns, Description of
the method and some applic
ations, cl in, chim, Acta, 5
: 52) 3-558.1960. ).

However, although the adsorbent in which heparin is immobilized on agarose shows selective adsorption ability for low-density lipoproteins, the adsorption ability is insufficient, and since agarose is used as a carrier, the mechanical strength is insufficient to handle it. It has poor performance and operability, and is prone to clogging when body fluids are poured into it.
The bore was destroyed during the sterilization process, making it extremely difficult to use.

Also, how to use glass powder and glass peas 1)
It had the drawbacks of low adsorption capacity and low adsorption selectivity, making it impractical.

In recent years, many attempts have been made to solve the above-mentioned problems and provide a low-density lipoprotein adsorbent that can be widely used, and patent applications have been filed (Japanese Patent Application Laid-Open No. 1983-1999)
-10243 (S, Patent Application No. 58-807781゜ (Problems to be Solved by the Invention) The content of the disclosure of the above invention is that the ability to adsorb low-density lipoproteins is completely insufficient to meet clinical requirements. It is thought to have low-density lipoprotein adsorption ability.

It is necessary to make it even higher and more practical.

Furthermore, it is preferable to use an adsorbent that adsorbs less polyvalent cations. We discovered that by using a polymer with sulfate groups on the surface of the adsorbent, the adsorption of polyvalent cations can be suppressed to a level that does not pose a clinical problem. We discovered that surprisingly high low specific gravity υ protein adsorption performance can be achieved only in a special range of ion exchange capacity and in a special narrow range of the adsorbent's ion exchange capacity. They discovered that when using a manufacturing method that involves bonding synthetic chain polymers, the above-mentioned adsorption ability can only be achieved in a special pH range of the solution of the synthetic chain polymers containing sulfate groups.

The present invention has now been completed.

That is, in the present invention, the molecular weight is 5×10′ or more.

A porous adsorbent having a size of 5 x 10' or less and a synthetic chain polymer portion having a sulfate group on the surface.

It is a low-density lipoprotein adsorbent characterized in that the ion exchange capacity of the adsorbent is 10 μeq/+l (wet capacity) or more and 500 μeq/ml (wet capacity) or less, and the pH of the solution (hydrogen ion concentration ) is 0.5 or more and 7 or less, a synthetic chain polymer solution having a sulfate group and an activated porous carrier are mixed. .

51 The target adsorbent of the present invention is low-density lipoprotein, and to explain it in more detail, it has a molecular weight of 2.2 x 1
06 to 3.5 x 106. Hydration density is 1.003 to 1. o 54 (r/l!tt), levitation coefficient (1,
065) from 0 to 20 x 10” as ・5ec-
'-dyn-1-r', diameter from 20.0 to 50. O
nm lipoproteins (5CANU A, M.

: plasma 1ipoproteins : a
n1ntroduction.

The Biochemistry of Ather
osclerosis” ed.

by 5CANU A, M, 1979. P. Go to 38. ). Lipoproteins with lower specific gravity, ie.

The levitation coefficient (1,0+S3) is 20X10-heavy” cl
Lipoproteins larger than s-5ec-'-dyn''''1·y-+ may be adsorbed, but high-density lipoproteins with high specific gravity are preferably not adsorbed.

In the present invention, the synthetic chain polymer portion having a sulfate group has a molecular weight of 5 x 10" or more and 5 x 10" or less, and 1
A chain polymer with many sulfate groups in the molecule, which is obtained by synthesis.

Examples include synthetic polymers such as polyvinyl sulfate, polystyrene sulfate, poly-2-acrylamide-2-methyl-1-propane sulfate, and copolymers containing these.

Synthetic polymers have excellent chemical stability and are easy to obtain that are stable against high-pressure steam sterilization, gamma ray sterilization, ethylene oxide sterilization, etc., and the degree of polymerization can be adjusted relatively easily. It is better than natural products and can be recommended. In addition, polymers obtained by synthesis are preferred because they can easily be obtained that do not easily cause complement activation as seen in natural polysaccharide polyanions. Furthermore, more favorable results can be obtained in that a polymer having a high degree of polymerization can be immobilized in a high retention amount on the phase, such as a vinyl polymer.

In addition, since low-density lipoprotein, which is the target substance for adsorption, is a huge lipoprotein with a diameter of about 20 OA, it is preferable that the structure of the synthetic polymer portion having a sulfate group is a chain structure, and it is long from the surface of the adsorbent. It is preferable that it is stretched. Further, it is preferable that the negative charge density in the polyanion moiety is at least one per 1 molecular weight of 500. More preferably, the number is one or more per molecular weight of 350, and one or more per molecular weight of 150.
It is desirable that there be one for every 250 units. The molecular weight here includes the molecular weight of the sulfate group.

Next, the molecular weight is an indicator of the length of a synthetic chain polymer molecule having a sulfate group, but if the molecular weight is less than 5 x 103, the length of the polymer molecule becomes short, and a sufficient amount of low-density lipolymer Proteins cannot be adsorbed. Conversely, if it exceeds 5X10', the molecule becomes too long and blocks the pores of the porous adsorbent, causing steric hindrance due to the synthetic chain polymer with sulfate groups, which reduces the ability to adsorb low-density lipoproteins. It goes down. The preferred molecular weight range is 1.75X10' to 3.5X10', more preferably 2.8X104 to 2.5X106,
Desirable size is 5 x 10' to 1.75 x 106
is within the range of Synthetic linear polymers with sulfate groups having molecular weights in this range exhibit good low-density lipoprotein adsorption capacity in porous adsorbents.

It is thought that the large number of sulfate groups possessed by the polymer moiety recognize multiple points on the low-density lipoprotein, thereby binding the low-density lipoprotein in a strong manner.

The ion exchange capacity of the low-density lipoprotein adsorbent of the present invention is determined when the molecular weight of the synthetic chain polymer molecule having a sulfate group is 5 x 1.
In the range from 0” to 5×101+, 10μeq/
i (wet capacity) to 600 μeq/+++/(wet capacity). Exceeding 300 μeq/ml means that the amount of synthetic chain polymer with sulfate groups is too large, which not only blocks the pores of the adsorbent and deteriorates the adsorption performance of low-density lipoproteins, but also Because the amount of negative charge is too large, non-selective adsorption increases, and the coagulation system, complement system, etc. are activated, which poses a problem when treating body fluids. Conversely, if the ion conversion capacity is 10μeq/
If it is smaller than ml, it means that the amount of synthetic chain polymer having sulfate groups is small, and the low-density lipoprotein adsorption performance will drop sharply. The preferred ion exchange capacity range is from 15 to 150 μe q/mz, more preferably from 20 to 100 μe q/ml, and more preferably from 25 to 100 μe q/ml.
The range is from 75 μeq/m.

= 9- The ion exchange capacity can be measured according to a normal method for measuring the ion exchange capacity of a cation exchange resin (eg, pH titration method).

The method for producing the adsorbent of the present invention includes, for example, activating the phase and covalently bonding a synthetic chain polymer having a sulfate group with a molecular weight of 5 x 10'' to 5 x 106; Examples include a method of graft polymerizing monomers to form a graft chain of a polymer having a sulfate group.

As for the shape of the carrier, any of the shapes such as spherical, granular, filamentous, hollow fiber, and flat membrane shapes can be effectively used, but spherical or granular shapes are particularly preferred from the viewpoint of flow of body fluid during extracorporeal circulation. Spherical or granular particles with an average diameter of 10 to 2500 μm are easy to use.

A range of 25 μm to 300 μm is preferably used.

The exclusion limit molecular weight (protein) of the carrier needs to be 2 million or more, preferably 2.5 million to 40 million, more preferably 3 million to 20 million.

Particularly preferable results are obtained when the total pore volume and pore size distribution of the phase are within the ranges described below.

The total pore volume and pore diameter of the phase are determined by mercury intrusion method (for example, Catalyst Engineering Course-4, Catalyst Measurement Method, Catalysis Society Cotton).

This refers to the value obtained by calculation from the mercury intrusion curve obtained from Jijinshokan, pages 69 to 73).

In the mercury intrusion method, the mercury pressure curve cannot be obtained unless the adsorbent is dried, so for adsorbents with drying shrinkage,
It is necessary to compensate for the shrinkage caused by drying. For example, if the volume shrinks to Vx" due to drying, the area becomes 1/x" and the diameter becomes 1/X, which means that the area is x3 times xt and the diameter becomes 1/X.

Correct by multiplying by X.

The total pore volume is preferably 0.5QC/S' or more,
1, occ7y or more is more preferable. It is preferably greater than 2.0cc/f, and preferably greater than 5.0cc/f.
It is more desirable that it be f or more. The pore capacity depends on the material, but the larger the value, the larger the internal space of the adsorbent per unit volume, and the greater the adsorption capacity for low-density lipoproteins.

As for the pore size distribution of the phase, it is preferable that 70% or more of the total pore volume l· is contained in the pore size range of 200× to 125 oon. That is, it is preferable that the pore size is widely distributed on the pore diameter side larger than the diameter of the low-density lipoprotein.

The pore size distribution state is that when the pore size is D, the pore capacity in the range of 0.8 D to 1.2 D is the total at any pore size (200; to 12 sooX). Less than 80% of the pore volume is required. That is, it is preferable that the pores are not concentrated only in a specific pore size range, but are distributed over a wide pore size range.

When attempting to adsorb low-density lipoproteins from blood or body fluids, it is desirable that the pores be concentrated in the pore size range of 200 to 300X in order to maximize the adsorption surface area of the low-density lipoproteins. , if the pore size distribution is narrow, very low-density lipoproteins (300-800^ in diameter) and chylomicrons (750-800^ in diameter), which have a larger diameter than low-density lipoproteins,
Coexisting substances such as 10,000λ) may cause clogging on the adsorbent particle surface. Once clogging occurs, low-density lipoproteins cannot enter the adsorbent particles, and the adsorbent's low density The ability to adsorb specific gravity lipoproteins decreases. In order to prevent clogging on the surface of adsorbent particles, K should use an adsorbent with a large pore size.

In this case, the surface area of the adsorbent becomes small, and the adsorption capacity for low-density lipoproteins becomes small.

In this way, an adsorbent with a narrow pore size distribution is extremely susceptible to the effects of coexisting substances in blood and body fluids.

It is very difficult to improve adsorption performance. On the other hand, in the case of adsorbents with a wide pore size distribution, very low density lipoproteins and chylomicrons, which have a diameter larger than that of low density lipoproteins, are captured in the large pores, so low density lipoproteins The pores through which the particles pass through are less likely to be crushed, and as a result, the adsorption capacity can be significantly increased.

A more preferable pore size distribution state is when the pore size is D,
For any pore size, the pore volume in the range of 0.8D to 1.2D is -15= 75% or less of the total pore volume, preferably 1470% or less, and more preferably 65
% or less.

The surface area of the carrier with a pore diameter of 250^ or more is determined from the positive size curve obtained by the mercury intrusion method, based on the assumption that the pores are uniformly cylindrical and do not intersect infinitely: va-b Sa-b=□-ra, -b Sa-b: Surface area between pore diameter a and pore diameter Va-1):
j Pore capacity ra-b:
It is an integral value of the pore diameter of 2501 or more of the surface area defined by the value calculated by the formula of average pore diameter,
The surface area S with a pore diameter of 250^ or more is expressed by the following formula.

S=,T,2. , 2/ r −D(r)drD (
r): Pore distribution function r: Radius of pore If this value is small, the adsorption surface area becomes small, and the adsorption ability of low-density lipoproteins decreases.

Preferable surface area (surface area with pore diameter of 250^ or more) Fi, 10rrL2 or more per adsorbent, more preferably 15 m
2 or more, preferably Id 20 m'' or more.

Due to the synergistic effect of a wide pore size distribution and a large surface area with a pore diameter of 250'A or more, the low-density lipoprotein adsorption ability of the polyanionic part is maximized, and high low-density lipoprotein adsorption performance can be obtained.

Since the adsorbent of the present invention is used for purification treatment of one body fluid, it is necessary that no clogging occurs when the body fluid is poured. Therefore, the phase used in the present invention is preferably a hard carrier, and synthetic polymer carriers, inorganic carriers, etc. are preferably used.

The hard carrier here refers to a carrier whose physical properties maintain a certain value or higher when an external force is applied to it. Specifically, gel is filled into a container with a diameter of 10 mm and a length of 50III+, and one container is used. When water is added, it is preferable that the volumetric shrinkage of the gel 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 1-j5%, and still more preferably 3% or less.

The carrier has a molecular weight of 5 x 10' or more, 5 x 10'
Any material may be used as long as it can fix the following synthetic chain polymer having sulfate groups. Supports that can be used include cellulose gel, Textran gel, agarose gel, vinyl polymer gel, polyacrylamide gel, polyhydroxyethyl methacrylate gel, organic or inorganic porous materials such as glass and silica, etc. 1 All carrier materials used in conventional affinity chromatography can be used.

Among the above-mentioned carriers, in particular, a phase composed of a crosslinked copolymer mainly composed of vinyl alcohol units has a small interaction with solutes such as proteins in plasma due to its hydrophilicity.
Reduce non-specific adsorption to a minimum. It has extremely excellent properties such as compatibility with plasma, does not interact with the complement system in plasma, and does not interact with the coagulation system. In terms of physical properties, it exhibits an excellent pore size distribution, has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers.

When used as a carrier for adsorbent for whole blood, it also has extremely excellent properties such as having little interaction with blood cell components and minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. .

A crosslinked polymer having vinyl alcohol units as a main component can be synthesized by polymerizing a monomer having a hydroxyl group or by introducing a hydroxyl group through a chemical reaction of the polymer. It is also possible to synthesize both in combination.

As the polymerization method, a radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or by chemical reaction between polymers (between polymers or between polymer and crosslinking agent), or both may be used in combination.

For example, '5=A&f' can be produced by copolymerization of a vinyl monomer and a vinyl or allyl crosslinking agent. In this case, vinyl monomers include vinyl acetate,
Vinyl esters of carboxylic acids such as vinyl gropionate, vinyl ethers Wi such as methyl vinyl ether, ethyl vinyl ether, etc. All examples can be listed.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as nitylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary.

In the case of vinyl copolymers, triallylisocyanurate of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a sulfinyl compound (allyl compound) having an incyanurate component and hydrolyzing the copolymer. The crosslinked material provides a particularly good phase in terms of strength and chemical stability.

Methods for immobilizing a synthetic chain polymer having a sulfate group with a molecular weight of 5 x 10' or more and 5 x 106 or less on the surface of an insoluble carrier include covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the polymer surface. Any known method can be used, but considering the elution properties of the immobilized compound, it is preferable to use it after covalent bonding, immobilization, or insolubilization.

For this purpose, we usually use immobilized enzymes, known methods for activating carriers used in affinity chromatography, methods for binding with ligands, and grafting in which the carrier or activated carrier is a backbone polymer and synthetic chain polymers having sulfate groups are used as branches. Polymerization techniques can be used.

Examples of activation methods include halogenated cyanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, 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 nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the ligand. However, in consideration of chemical stability, thermal stability, etc., a method using an epoxide is preferable, and an epichlorohydrin method is particularly recommended.

For carriers with silanol groups such as silica and glass, γ-glycidoxyglobiltrimethoxydisilane, γ-aminoglopyltriethoxysilane, γ-mercaptoglobiltrimethoxysilane, vinyltrichlorosilane, etc. are still available. Various silane coupling agents are preferably used.

The bonding reaction between the activated carrier and the synthetic chain polymer having a sulfate group depends on the type of functional group on the activated carrier side and the functional group on the synthetic chain polymer side having a sulfate group, reaction temperature, time,
Optimal conditions such as pH are selected, but in the case of synthetic chain polymers with sulfate groups, the inventors have found that the pH of the solution of synthetic chain polymers with sulfate groups has a very important meaning. revealed by research. The p)I of the synthetic chain polymer solution with sulfate groups recommended by the present inventors is (], 5
to 7, but when the pH is lower than 0.5,
Because the polymer molecules form a rigid string and are fixed to the phase as they are, the polymer molecules bound to the carrier cannot freely extend even in body fluids, and function as a synthetic chain polymer with sulfate groups. Since the amount of bound polymer is still too large, the adsorption ability of low-density lipoprotein becomes low. Also, p
When H is higher than 7, the polymer molecules stretch sufficiently in the solution and become a so-called hard polymer, so the polymer cannot penetrate sufficiently into the network structure of the phase, and the amount of bonded polymer becomes absolutely small. However, the adsorption amount of low-density lipoproteins decreases. The pH recommended by the inventors is
In the range of 5 to 7, the synthetic chain polymer molecules having sulfate groups are in a gently spread state, so that the bonding to the carrier is also carried out in a moderately spread state. Therefore, even when the adsorbent is immersed in body fluids, the bound polymer molecules stretch gently, allowing it to fully function as a synthetic chain polymer with sulfate groups, increasing its ability to adsorb low-density lipoproteins.

The preferred pH range is 1 to 6, more preferably 1.5 to 5, and desirably 2 to 4.

Next, examples of graft polymerization methods include methods that utilize chain transfer reactions, methods that utilize reactions such as dehydrogenation and dehalogenation by radiation, ultraviolet light, etc., and methods that utilize the formation of peroxides. A method in which a monomer having a sulfate group is graft-polymerized onto a phase having a reducing group such as a hydroxyl group, a thiol, an aldehyde, or an amine using a cerium salt, an iron salt, or the like as an initiator is simple and recommended.

In addition, a graft polymerization system is preferably used because a polymer having a relatively large molecular weight can be fixed to the inside of the phase.

The above is an example of the method for manufacturing the adsorbent of the present invention, and the molecular weight is 5.
Although the method of bonding synthetic chain polymers having sulfate groups of X 10s to 5 X 10' has been described in detail, the present invention is not limited thereto.

For example, a method of polymerizing (copolymerizing) using a polymerizable monomer or a crosslinking agent having a synthetic chain polymer moiety having a sulfate group with a molecular weight of 5 X 10S to 5 X 10'', -22 = crosslinked polymer particles further At the time of post-crosslinking, a method using a crosslinking agent having a synthetic chain polymer portion having a sulfate group can also be used.Also, a method in which a synthetic chain polymer having a sulfate group is activated and then bonded to a carrier. can also be adopted.

The adsorbent of the present invention is generally used while being filled in a container equipped with an inlet and outlet for body fluids.

In Figure 1, 1) 1''1). This shows an example of an adsorption device containing the low-density lipoprotein adsorbent of the present invention. Cap 6 having body fluid inlet 5 through backing 4
A cap 8 having a body fluid outlet lower part is screwed into the opening at the other end of the cylinder 2 through a backing 4' with a filter 6' stretched inside to form a container. The adsorbent layer 9 is formed by filling and holding an adsorbent in the gap 5'.

The adsorbent layer 9 may be filled with the low-density lipoprotein adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. Other adsorbents that can be used include, for example, activated carbon, which has a wide range of adsorption capacities. Due to the synergistic effect of this adsorbent, a wide range of clinical effects can be expected.

The volume of the adsorbent layer 9 is 50 to 4 when used for extracorporeal circulation.
Approximately 0-0 is appropriate. When the device of the present invention is used for extracorporeal circulation, there is a two-part method as described by Ohji. First, blood taken from the body is separated into plasma components and blood cell components using a centrifuge or a model plasma separator, and the plasma components are passed through the device to be purified and then separated into blood cell components. One method is to return the blood to the body along with the blood, and the other method is to pass the blood taken out from the body directly through a nuclear device and purify it.

In addition, regarding the passage speed of blood and plasma, the adsorption efficiency of the adsorbent is very high, so the particle size of the adsorbent can be coarsened, and the amount of packed dust can be reduced, so the shape of the adsorbent layer can be adjusted. A high passing speed can be provided regardless of the method. Therefore, a large amount of body fluid can be treated.

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

(Effects of the Invention) As described above, the low-density lipoprotein adsorbent of the present invention selectively removes and adsorbs low-density lipoproteins in body fluids at a high rate, and an adsorption device using the adsorbent can: It is extremely compact, convenient and safe.

Using a porous adsorbent in which a specific amount of synthetic chain polymers with sulfate groups with a specific degree of polymerization are present on the surface, it has a much higher ability to adsorb low-density lipoproteins than conventional ones. It has become a low-density lipoprotein adsorbent that has the following characteristics and hardly adsorbs polyvalent cations.

Furthermore, since the ion exchange capacity was suppressed to 300 μeq/nt (wet capacity) or less, there was little non-selective adsorption and less activation of the coagulation fibrinolytic system and the complement system, making it a safe adsorbent.

Furthermore, by using the method for manufacturing the low-density lipoprotein adsorbent of the present invention-25, the above-mentioned adsorbent can be easily obtained, and it has become possible to stably obtain the low-density lipoprotein adsorbent.

The present invention can be applied to general methods of purifying and regenerating body fluids such as hyperlipidemia, and is effective for safe and reliable treatment of diseases caused by hyperlipidemia.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Hinyl acetate 1005',) realyl isocyanurate 4
L4 y, ethyl acetate 100S', heptane 1002
, polyvinyl acetate (degree of polymerization 500) 7.51 and 0
2. A homogeneous mixture of z'-azobisisobutyronitrile 5.82%, polyvinyl alcohol 1% by weight, sodium dihydrogen phosphate dihydrate o, oslIJJ 1% and disodium hydrogen phosphate dodecahydrate After 400% of water dissolved in 1.5% by weight was placed in a flask and thoroughly stirred, suspension polymerization was carried out by heating and stirring at 155C for 18 hours and then at 75C for 5 hours to obtain a granular copolymer. During the polymerization, the stirring speed was controlled to keep the particle size small.

After washing with Oki water and then extracting with acetone, add 46% of caustic soda.
40 ip in a solution of 5 F and 2 T methanol.
The transesterification reaction of the copolymer was carried out for 18 hours.

The average particle size of the obtained gel was 404 ml, and the vinyl alcohol units (qOH) per unit weight were 9. Omeq/? The specific surface area was 100 m'', the molecular weight exclusion limit for proteins and viruses was 2 x 107.The obtained gel was used as a carrier for an adsorbent.

The resulting gel 102 (dry weight) was suspended in 120 Tnl of dimethyl sulfoxide, and epichlorohydrin 78°3-150% sodium hydroxide 10-1 was added thereto.
The activation reaction was carried out with stirring at DC for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. The epoxy equivalent of the activated support is 120 μeq/m
l (wet capacity).

Next, polyvinyl potassium sulfate (molecular weight 2.43X10
') 1015mg' (1) - Diluted to 50% with distilled water and used as a ligand solution. pH is 3. with hydrochloric acid. Adjusted OK. Add the activated carrier 10- to this ligand solution,
Ligand binding reactions were carried out for 16 hours at p.

Thereafter, the gel was transferred onto a glass filter, washed with a sufficient amount of water, and dehydrated by suction.

Next, in order to make the obtained adsorbent into Na type, the above adsorbent was placed in 100% of 0.1N sodium hydroxide solution,
Shake for 30 minutes at room temperature.

Thereafter, a sufficient amount of water was washed to obtain a Na-type adsorbent.

When the ion exchange capacity of this adsorbent was measured, it was found to be 40μ
eq/m (wet capacity).

The ion exchange capacity was measured as follows.

First, in order to convert the Na-type adsorbent into H-type, the Na-type adsorbent 5- was added to 0.1 N hydrochloric acid 5-, and after shaking at room temperature for 30 minutes, it was thoroughly washed with distilled water. Next, after being dehydrated by suction, it was suspended in 60 g of distilled water, and a pH titration curve was determined using 0.1N sodium hydroxide. The ion exchange capacity was calculated from the pH curve obtained as described above.

Next, the ability of the Na-type adsorbent to adsorb cholesterol was investigated using plasma from patients with familial hypercholesterolemia.

In the adsorption experiment, 12-
Plasma of a patient with familial hypercholesterolemia was added and incubated at 370°C for 3 hours with shaking. After incubation, the adsorbent was allowed to settle and the supernatant was analyzed and compared to the patient plasma used.

Total cholesterol (hereinafter abbreviated as TC) was analyzed using the enzymatic method, high-density lipoprotein (hereinafter abbreviated as HDL-C) by the heparin-manganese precipitation method, albumin by the bromcresol green method, and fibrinogen by the single-method. Calcium was measured using the radial 6 immuno-auto-diffusion method and the orthophresolphthalein combination color method.

Here, in the case of familial hypercholesterolemia patient plasma,
The total cholesterol value is mostly dominated by cholesterol in low-density lipoproteins (hereinafter abbreviated as LDL-○), and cholesterol in high-density lipoproteins and very-low-density lipoproteins accounts for a very small proportion. few. therefore,
A decrease in total cholesterol approximately means a decrease in low density lipoprotein.

As a result of analysis, TC in plasma was 41C1η/ml, while adsorption fit tri was 160 mg/ml.
tK decreased. i.e. 1-equivalent of adsorbent, 60 mg
adsorbed cholesterol. On the other hand, HDL-C
is 25rng/ml1. 2) my/mlt (91% before adsorption), albumin is 3.5 g/ml is 3.4
f/mlt (97%), fibrinogen is 190
rn9/mlt is 170mQ/mlt (a9e), calcium is 4.5mEq/l 4,3 rnEQ//
, '(96%), which hardly decreased, and T C(:LDL
-C) was selectively adsorbed at a high rate.

-6- Comparative Examples 1 to 2 and Examples 2 to 9 Using the same carrier as in Example 1, it was activated with epichlorohydrin in the same manner as in Example 1, and polyvinyl potassium sulfate of various molecular weights were bound. The concentration of the potassium polyvinyl sulfate solution was kept constant (2.03 t/ml) regardless of the degree of polymerization, and the ligand binding reaction was carried out in the same manner as in Example 1.

The molecular weight of the polyvinyl potassium sulfate used was 3.5.
X 103.5 X 103.2.2 X 10', 9
X 10', 2.4 X 105.8 X 10',
5 x 10', 7. I x 10'. Among these, the molecular weight is 5.5 x 103 and 7.1 x 101! These two types were used as comparative examples.

After the binding reaction of the ligand was completed, it was made into Na type in the same manner as in Example 1, and the adsorption capacity was measured. The results are shown in FIG. In Figure 2, the horizontal axis shows the molecular weight of polyvinyl potassium sulfate used as a ligand, and the vertical axis shows the amount of cholesterol adsorbed by the adsorbent. It can be seen that a cholesterol adsorption amount of 20 m97 m1 (wet capacity) or more can be achieved only within the range of '. In addition, the ion exchange capacity of all adsorbents is 10 to 300 μeq/++/
(wet capacity).

Comparative Examples 3 to 4 and Examples 10 to 14 The same phase as in Example 1 was used, activated with epichlorohydrin in the same manner as in Example 1, and polyvinyl potassium sulfate having a molecular weight of 2.4 x 105 was bound to the carrier at various concentrations. Ta.

The concentrations of potassium polyvinyl sulfate were 0.2 and 0.4.

1.0, 2.0, 2.5, 4.0, 6. Of/
mll-, and in the same manner as in Example 1, activated carrier 10 m/
Ligand binding reaction was carried out at a ratio of 50 to 50% of potassium polyvinyl sulfate solution.

After the completion of the ligand binding reaction, the Na type was used in the same manner as in Example 1, and the ion exchange capacity was measured.As a result, the ion exchange capacity was 5, 10, 2, from the lowest to the highest concentration of polyvinyl potassium sulfate.
It was 0,40,70,280°3401ieq/ml (wet capacity). Here, 5.340 μeq/ml is a comparative example.

Next, in the same manner as in Example 1, an adsorption test of the Ha type adsorbent was conducted. The results are shown in FIG. 6, where the horizontal axis shows the ion exchange capacity of the adsorbent, and the vertical axis shows the amount of cholesterol adsorbed.

From this result, the ion exchange capacity of the adsorbent is 10 to 500.
It can be seen that good cholesterol adsorption ability is exhibited only in the μeq/ml (wet volume) range.

Comparative Example 5 to 7 and Example 15 to 19 Same as Example 1, using the same phase as Example 1.

Activated with epichlorohydrin, molecular weight 2.4 x 101
) was bound to the carrier at various pHs (hydrogen ion concentrations).

The ligand solution was prepared in the same manner as in Example 1, and then its pH was adjusted.

The pH is determined using hydrochloric acid or hydroxide solution, and is 0.3.0.5.1, 2, 3, 5, 7, 9.12.
A ligand solution adjusted to Here, pH 0.5
, 10, and 12 are comparative examples.

All other conditions were the same as in Example 1 to synthesize the adsorbent,
When we investigated its cholesprol adsorption performance, the results were as shown in Figure 4. In Figure 4, the horizontal axis shows the p of the ligand solution.
H1 The vertical axis shows cholesprol adsorption ability.

This result shows that good cholesterol adsorption ability can be exhibited only when the pH is within the range of 0.5 to 7.

Still, the ion exchange capacity at this time is 560, 280, 120, 60°40.25, 15° in order from the lowest pH.
, 7.4, and when graphed with pH as the horizontal axis, we get
It will look like Figure 5.

Further, when the horizontal axis is the ion exchange capacity and the vertical axis is the cholesterol adsorption performance, the result is as shown in FIG. 6.

This graph also shows that the ion exchange capacity is 10 to 300 μe.
It can be seen that high cholesterol adsorption performance can be exhibited only within the range of Q/ml.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing an example of a low-density lipoprotein adsorbent containing the low-density lipoprotein adsorbent of the present invention, and FIG.
The figure is a graph showing the experimental results of Examples 2 to 9 and Comparative Examples 1 to 2, and Figure 3 is a graph showing the experimental results of Examples 2 to 9 and Comparative Examples 1 to 2.
4 to 6 are graphs showing the experimental results of Examples 15 to 19 and Comparative Examples 5 to 7.

Claims (2)

[Claims] (1) A porous adsorbent having a molecular weight of 5 x 10^3 or more and 5 x 10^6 or less and having a synthetic chain polymer portion with a sulfate group on its surface, the ion exchange capacity of the adsorbent being 1
0 μeq/ml (wet capacity) or more, 300 μeq/ml
(wet capacity) or less. (2) pH (hydrogen ion concentration) of the solution is 0.5 or more, 7
The molecular weight is 5 x 10^3 or more and 5 x 10^6 or less, and sulfuric acid A porous adsorbent having a synthetic chain polymer portion having a group on its surface, the adsorbent having an ion exchange capacity of 10 μeq.
/ml (wet capacity) or more and 300 μeq/ml (wet capacity) or less.