JPS60114340A - Adsorbent for adsorption of low specific gravity lipoprotein - Google Patents

Abstract

PURPOSE:To enable adsorption of low specific gravity lipoprotein with high efficiency by using an adsorbent which has the polyanion part having many functional groups exhibiting negative charge in a molecule and having a relatively large mol. wt. on the surface and of which the negative charge density is in a specific ragne. CONSTITUTION:The polyanion part having >=600mol.wt. (e.g., polyacrylic acid) is provided on the surface. The polyanion part of which the negative charge is >=1mueq/ml and <=1meq/ml and the mol. wt. is >=600 is made into the polyanion part having at least one functional group (e.g., carboxyl group and sulfonate group) exhibiting negative charge for each 300mol.wt. Such adsorbent adsorbs selectively the low specific gravity lipoprotein in a bodily fluid with high efficiency, decreases nonselective adsorption, has safety, permits simple sterilization operation and is suitable for cleaning up or regeneration of the bodily fluid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a low-density riboprotein adsorbent that selectively adsorbs and removes low-density riboproteins, which are thought to be closely related to various diseases caused by an increase in plasma lipids.

As is well known, an increase in blood lipids, particularly low-density riboproteins, is thought to be closely related to the cause or progression of arteriosclerosis. When arteriosclerosis develops, there is a very high possibility of developing serious circulatory system symptoms such as axillary cerebral infarction, and the mortality rate is also high.

Therefore, by selectively adsorbing and removing low-density riboproteins from body fluid components such as blood and plasma, it is expected that the progression of the above-mentioned diseases can be prevented, symptoms can be alleviated, and furthermore, healing can be accelerated. Ta.

Existing techniques that can be used for the above purpose include adsorption using adsorbents in which heparin is immobilized on agarose gel (Lupie
n, P-J, et, a4: A new ap
proach t.

the management of family
l hypercholesternlemia.

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

2:1261-1264, 1976), and chromatography using glass powder or glass beads (Carlson, L.A.: Chromatography
atographic separation of s
erum 1ipoprotein on glass
sprayer columns + Description
on of the method and some
applications, Cl1n, Chim, Ac
ta, 5:528-538.1960. ).

However, although the adsorbent in which heparin is immobilized on agarose shows selective adsorption ability for low-density riboproteins, its adsorption ability is insufficient, and because it uses agarose as a carrier, it has insufficient mechanical strength. It is difficult to handle and operate, and can easily cause eyelids when body fluids are poured into it.
The bore was destroyed during the sterilization process, making it difficult to use.

In addition, the method using glass powder or glass peas is
It had the drawbacks of low adsorption capacity and low adsorption selectivity, making it impractical.

In view of the problems of adsorbents based on the prior art as described above, an object of the present invention is to be generally applicable, to selectively adsorb low-density riboproteins with high efficiency, and to minimize non-selective adsorption. The present invention aims to provide an adsorbent that is safe, easy to sterilize, and suitable for body fluid purification or regeneration.

As a result of intensive research in line with the above objectives, the present inventors discovered that the molecule has many functional groups exhibiting negative charges, has a relatively large polyanion moiety on its surface, and has a negative charge density. Adsorbents in a specific range adsorb low-density riboproteins with surprisingly high efficiency, exhibiting less non-selective adsorption and less activation of blood coagulation, fibrinolytic system, and complement system. This discovery led to the completion of the present invention.

That is, the present invention has a polyanion portion having a molecular weight of 600 or more on the surface and a negative charge density of 1 μe.
It is an adsorbent for adsorbing low-density riboproteins, which is characterized by having a molecular weight of 60 meq/- or more and 1 meq/- or less.
It is preferable that the polyanion moiety having a molecular weight of 0 or more is a polyanion moiety having a chain structure, and the polyanion moiety having a molecular weight of 600 or more is preferably at least 1 per molecular weight 300.
It is preferable that the polyanionic moiety is a polyanionic moiety having a functional group exhibiting negative charges.

The target adsorbent of the present invention is low-density riboprotein, and to explain in more detail, it has a molecular weight of 2.2
0' to 5,5
is 0 to 20 X 1 o-"crn'5ec-" d
yn-'-? -', diameter from 20.0 to SO, Onm
riboproteins (5CANU, A, M.

: plasma 1ipoproteins : an
1ntroduction.

'The Biochemistry of Ath
erosclerosis” ed.

by 5CANU A8M, 1979, P, 5
~8. ).

Riboproteins with a smaller specific gravity, i.e., levitation coefficient (
1,063) is 20 X 10-"crn-8ec-'
Although riboproteins larger than -dyn-' j f' may be adsorbed, it is preferable that high-density riboproteins with high specific gravity are not adsorbed.

The polyanionic moiety referred to in the present invention means that the molecular weight of one molecule is 6.
00 or more and has a negative charge in one molecule, that is, a carboxyl group (C0OH).

coo-), sulfonic acid groups (So, H, 5os-), and a large number of functional groups that exhibit negative charges in plasma.

Examples include polyacrylic acid, polyvinylsulfonic acid,
Vinyl synthetic boria 5-ions such as polyvinyl phosphate, styrene polyanions such as polystyrene sulfonic acid and polystyrene phosphate, peptide polyanions such as polyglutamic acid and polyaspartic acid, RNA,
Examples include polyanions based on nucleic acids such as DNA, polymethacrylic acid, polyphosphoric acid, polyphosphate ester, poly-α-methylstyrene sulfonic acid, and styrene-maleic acid copolymers.

Among them, polyanions obtained by synthesis are
It has excellent chemical stability, and is easy to obtain that is stable against high-pressure steam sterilization, gamma ray sterilization, ethylene oxide sterilization, etc., and the molecular weight can be adjusted relatively easily. Better than anything else and can be recommended. In addition, polyanions obtained by synthesis are preferable because polyanions that do not easily cause complement activation as seen in natural polysaccharides can be easily obtained. Furthermore, products such as vinyl anions that can be directly subjected to craft polymerization on a carrier are better in that polyanions with large molecular weights can be immobilized on the carrier in a high retention amount. give.

In addition, since the low-density riboprotein, which is the substance to be adsorbed, is a huge riboprotein with a diameter of about 200X, it is preferable that the structure of the polyanion part is a chain structure, and it extends long from the surface of the adsorbent. It's better to be there. Further, it is preferable that the polyanion moiety has at least one negative charge density per 300 molecular weight. More preferably, the molecular weight is 2
The number is one or more per 00, and preferably one per unit of molecular weight 70 to 150. The molecular weight referred to here includes the molecular weight of a functional group that exhibits a negative charge. The molecular weight of the polyanion moiety needs to be at least 600 because as it becomes smaller, low-density riboproteins will not be adsorbed much. Preferably 5000 or more, 25000 to 10000
A range of 00 is desirable.

The polyanion moiety has a large number of negatively charged functional groups,
It is thought that by recognizing multiple points on low-density riboproteins, it binds low-density riboproteins with strong Coulomb force.

The density of negative charge is 1 μeq to 1 meq per adsorbent.
This range is an appropriate range that has good adsorption performance for low-density riboproteins, good adsorption selectivity, and little influence on the coagulation fibrinolytic system and the complement system. 1μeq/fn1. When the negative charge density becomes lower, the adsorption ability of low-density riboproteins falls short of practical performance, and when it exceeds 1 meq, non-selective adsorption increases, which adversely affects the coagulation fibrinolytic system and the complement system. A more preferred range is 5 μeq/rrt to 700 μeq/rnl, even more preferably 10 μeq/- to 500 μeq/m, and even more preferably 20 μeq/- to 300 μeq/ml.
It is.

The negative charge density can be measured according to a conventional method for measuring the ion exchange capacity of a cation exchange resin.

Methods for creating the adsorbent of the present invention include, for example, activating a carrier and covalently bonding a chain synthetic polyanion at one end thereof, graft polymerizing an anionic monomer to the carrier,
Examples include a method of forming a polyanion graft chain.

The carrier is a polyanion having a molecular weight of at least 6000 with a negative charge density of 1 μsq/- to 1 meq/TnI! .

Both hydrophilic and hydrophobic carriers can be used; however, when using a hydrophobic carrier, non-specific adsorption of albumin to the carrier sometimes occurs, so a hydrophilic carrier is preferable. Give results.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but at least 600
Particulate or fibrous forms are preferable in terms of the amount of polyanions having a molecular weight of 0 retained and ease of handling as an adsorbent.

The particulate carrier has an average particle size of 25 μm to 2500 μm.
Preferably, it is in the μm range. The average particle size is JIS-
After classification in running water using a sieve specified in Z-8801, the intermediate value between the upper limit particle size and the lower limit particle size of each class is taken as the particle size of each class, and the average particle size is calculated as the weight average. Further, the particle shape is preferably spherical, but is not particularly limited. If the average particle size is 2500 μm or more, the adsorption amount and adsorption rate of low-density riboprotein will decrease;
Below, activation of the coagulation system and blood cell adhesion are likely to occur.

9- Particulate carriers that can be used include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, silica-based activated carbon-based, etc., but hydrophilic carriers having a gel structure give good results. Furthermore, known carriers commonly used for immobilized enzymes and affinity chromatography can be used without any particular limitations.

Here, the protein exclusion limit molecular weight of the carrier needs to be 2 million or more, preferably from 2.5 million to 10 million, and preferably from 3 million to 7 million.

This is expressed in terms of the average pore diameter of 200° and 300°.
'A, more preferably in the range 250X to 700X, preferably in the range oo to 650X.

Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention can immobilize a polyanion having a molecular weight of at least 600 at a negative charge density in the range of 1 μsq/m/ to 1 meq/−, and the polymer composition is polyamide-based, Known polymers that can have a porous structure, such as polyester, -10-polyurethane, and vinyl compound polymers, can be used; however, vinyl compound porous polymer particles made hydrophilic with a hydrophilic monomer are particularly useful. give favorable results.

Since the adsorbent of the present invention is used for purification treatment of body fluids, it is necessary that no clogging occurs when body fluids are passed through it. Therefore, the carrier 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. Specifically, gel is filled into a container with a diameter of 10+n+i and a length of 50 mTA, and the gel is passed through the container. When watering, the volume reduction rate of the gel is 1 when the difference between the inlet pressure and outlet pressure of the column is 200 ml x Hg.
It is preferable that the coefficient is 0 or less.

The porous structure has an average pore size! 20OA to 5ooo'
It is preferable that the average pore size is within the range of A, and if the average pore diameter is too small, the amount of low-density lipoprotein adsorbed will be small;
If it is too large, it is not practical because the strength of the polymer particles decreases and the surface area decreases. The surface area is preferably at least 10 g/y, and is 55,771″
It is more preferable that the value is /li' or more, and b0 is preferably 1 (10g/7 or more).

The average pore diameter was measured using a mercury intrusion porosimeter.

In this method, mercury is injected into a porous material, and the pore size is determined from the amount of mercury intruded and the pore size is calculated from the pressure required for the intrusion. Pores of 40'5L or more can be measured. The pores of the present invention are defined as pores communicating from the surface with a pore diameter of 40 or more. The average pore diameter is dv/dl, where r is the pore diameter and ■ is the cumulative pore volume measured with a porosimeter.
Let it be the value of r when the directness of ogr is maximum.

When a fibrous carrier is used, the fiber diameter is preferably in the range of 1 μm to 50 μm, more preferably 5 μm to 25 μm. If the fiber diameter is too large, the adsorption amount and adsorption rate of low-density lipoproteins will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging are likely to occur.

The vine fibrous carrier used includes regenerated cellulose fibers,
Generally known fibers such as nylon, acrylic, and polyester can be used.

Methods for immobilizing a polyanion having a molecular weight of at least 600 on the surface of an insoluble carrier include covalent bonding, ionic bonding,
Any known method such as physical adsorption, embedding, or insolubilization by depositing on the surface of a polymer can be used, but considering the elution property of the polyanion, it is preferable to use the polyanion after fixation and insolubilization by covalent bonding. For this purpose, it is possible to use an immobilized enzyme, a known carrier activation method used in affinity chromatography, a binding method with a ligand, and a graft polymerization method in which the polymer and carrier are used as a trunk polymer and polyanions are used as branches. .

Examples of activation methods include cyanide rhodanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformer) method, 1,1'-carbonyldiimidazole method, etc. I can give it to you. The activation method of the present invention = 13- is limited to the above examples as long as it can perform a substitution and/or addition reaction with a nuclear reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, a thiol group, etc. 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.

Furthermore, various silane coupling agents are preferably used for carriers having silanol groups, such as silica-based and glass-based carriers.

Examples of graft polymerization methods include chain transfer method, emulsion polymerization method, graft polymerization method using various initiators such as cerium salt, persulfate-no-lithium chloride, hydrogen peroxide-Kinki salt, etc.
Graft polymerization methods using polymers having functional groups such as perester groups, mercapto groups, and diazo groups, graft polymerization methods using air or ozone oxidation, and radiation grafting methods include, among others, hydroxyl groups, thiols, aldehydes,
A method in which an anionic monomer is graft-polymerized onto a carrier having a reducing group such as an amine using a cerium salt, iron salt, or the like as an initiator is simple and recommended.

In addition, a graft polymerization system is preferably used because a polyanion having a relatively large molecular weight can be fixed inside the carrier.

Two or more types of polyanions having a molecular weight of at least 600 may be bound to the carrier.

The above is an example of the method for manufacturing the adsorbent of the present invention, and the polyanion having a molecular weight of at least 6000 is 1 μeq/mj!
Although the method of binding to a carrier with a negative charge density of 1 meq/ml has been described in detail, the present invention is not limited thereto.

For example, a method of polymerizing (copolymerizing) using a polymerizable monomer having a polyanion moiety having a molecular weight of at least 6000, a method of binding to a carrier after fully activating a polyanion having a molecular weight of at least 600, etc. can also be adopted.

That is, in the present invention, at least 600
Polyanion part with 0 molecular weight from 1μeq/- to 1m
The effect is exhibited by having a negative charge density of eq/c, and is not dependent on the manufacturing method.

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

In the drawings, reference numeral 1 shows an example of an adsorption device containing the low-density riboprotein adsorbent of the present invention, in which a backing 4 with a filter 3 placed inside is placed in an opening at one end of a cylinder 2.
Screw-fit the cap 6 having a body fluid inlet through the
A cap 8 having a lower body fluid outlet through a backing 4' having a filter 3' placed inside the opening at the other end of the cylinder 2.
are screwed together to form a container, and filters 5 and 3' are screwed together to form a container.
The adsorbent layer 9 is formed by filling and holding an adsorbent in the gap.

The adsorbent layer 9 may be filled with the low-density riboprotein 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. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The volume of the adsorbent layer 9 is, when used for extracorporeal circulation,
50~4007! The degree is appropriate. When the device of the present invention is used for extracorporeal circulation, there are two methods as follows: 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 blood taken from the body directly through a nuclear device and purify it.

In addition, since the adsorption efficiency of the adsorbent is very high when the blood or plasma passage rate is 9, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. It is possible to give an equally high passing speed. Therefore, a large amount of body fluid can be treated.

The method for passing body fluids may be continuous, depending on clinical needs or equipment conditions.
It is also possible to use the liquid intermittently.

As described above, the adsorbent of the present invention selectively adsorbs and removes low-density riboproteins in body fluids at a high rate, and an adsorption device using the adsorbent is extremely compact, simple, and safe. It is. In addition, other components in plasma proteins are less likely to be adsorbed non-selectively, low-density riboproteins can be adsorbed with high efficiency, and coagulation fibrinolysis and complement system activation are less likely to occur.

The present invention is applicable to general usage for purifying and regenerating body fluids such as hyperlipidemia, and is effective in safely and reliably treating diseases caused by hyperlipidemia.

Embodiments of the present invention will be explained in more detail with reference to Examples below.

Example 1 Graft polymerization of acrylic acid was carried out using Sepharose 4B (manufactured by Pharmacia, Sweden) as a carrier. First, add 1 part of Sepharose 4B to 200rrLt of distilled water.
0- (wet capacity), followed by nitrogen substitution, then 35 mmot of acrylic acid (having a molecular weight of 72 and -18-1 carboxyl group) was added.
Finally, 2.5 mmot of ceric ammonium nitrate was dissolved in 50 rnt of 0.1N nitric acid and added. Thereafter, graft polymerization was carried out under a nitrogen atmosphere at 35C for 3 hours with stirring. After the reaction, the mixture was thoroughly washed with water to obtain an adsorbent for adsorbing low-density riboproteins. The ion exchange capacity, ie, negative charge density, of the obtained adsorbent was measured by a conventional method and found to be 40 μeq/+++z.

In the adsorption experiment, 10 volumes of hyperlipidemic patient plasma and 1 volume of adsorbent were mixed and incubated at 37C for 5 hours with shaking. After adsorption, low-density riboproteins (hereinafter abbreviated as LDL) were extracted by the heparin-calcium precipitation method, high-density riboprotein cholesterol (hereinafter abbreviated as HDLch) by the heparin-manganese precipitation method, and albumin by the bromcresol green method. Measured at The analysis results showed that LDL in plasma was 704 my/d! On the other hand, after adsorption it decreased to 34 s my/di (49% before adsorption (7)), but HDLch decreased to 17fnQ/di and 17fn9.
7 dl (100%), albumin is 3.5? /dl
Ka3.51il/d! (100%) and does not fall, L
DL was selectively adsorbed.

Example 2 Vinyl sulfonic acid (molecular weight 108
A low-density riboprotein adsorbent was obtained by carrying out graft polymerization in the same manner as in Example 1, except for using a sulfonic acid group (having one sulfonic acid group per sulfonic acid group). The ion exchange capacity, that is, the negative charge density, of the obtained adsorbent was measured by a conventional method and was found to be 52μ.
eq/1nt. The adsorption experiment was conducted in the same manner as in Example 1. As a result, LDL was 704 rR97dlte6t&(7)K, but after adsorption it decreased to 310~/di (44 di before adsorption), but HDLch was 17 m97
di is 17 m9/di (1o.

H), albumin is 3.5 t/di and 3.4 t/d
i (97%), which hardly decreased.

Comparative Example 1 Graft polymerization was carried out in the same manner as in Example 1, except that the amount of ceric ammonium nitrate was changed to 0.25 mmoj. The ion exchange capacity of the obtained adsorbent was 0.7 μeq/−.

When an adsorption experiment was conducted in the same manner as in Example 1, LDL was 704 my/dl and 6 a3 mg/dz (97
%), HDLch is 17 h19/dl is 17 my/
cit (1o o %), albumin is 3.5 Y
/di was 3.5 f/dt (1 o %), and almost no adsorption was observed.

Example 3 Vinegar mVyl 1o o y,) Reallylisocyanurate) 41.4fI (X=0.40), Ethyl acetate 1o.

v1 heptane 100 f, polyvinyl acetate (polymerization degree 50
0) 7.5? and 3.81% of 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate and 12% by weight of disodium hydrogen phosphate. Add 4,001 nt of water in which 1.5% by weight of hydrate was dissolved and stir thoroughly.
Suspension polymerization was carried out by heating and stirring at 5c for 5 hours to obtain a granular copolymer. After filtration, washing with water, and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.59 g of caustic soda and 2 t of methanol at 40 °C for 18 hours.

The average particle size of the obtained gel was 150 μm, and the vinyl alcohol units (q OH) per unit weight were q, o m
eq/f, specific surface area was 60rrt/f, and exclusion limit molecular weight by dextran was 6 x 10'.

Using the obtained gel as a carrier, this carrier 10 was immersed in 200 mm of distilled water and replaced with nitrogen, and then 15 mmol of ceric ammonium nitrate was added to 50 mm of 0.1N nitric acid.
t was dissolved and added. After reacting for 20 minutes at 35C under a nitrogen atmosphere, the sample was washed with water and immersed in 200ml of distilled water to which 70 mmol of acrylic acid had been added. Thereafter, a reaction was carried out at 35C for 3 hours under a nitrogen atmosphere with stirring. After the reaction, it was thoroughly washed with water to obtain an adsorbent for adsorbing low-density riboproteins. When the ion exchange capacity of the obtained adsorbent was measured, it was found that 2
It was 20μeq/-.

The adsorption experiment was conducted in the same manner as in Example 1. As a result, L
DL dropped to 7041V/#282my/di (40ch), while HDLch was 17m97di7ri x161n9/di (94%), and albumin was 5.5
17di-22- hardly decreased to 3.4 t/di (97%).

Comparative Example 2 A cation exchange resin IRC-50 with C0OH group manufactured by Rohm & Haas (ion exchange capacity 3.0meq/ml, pore size 2110-21]00X, particle size 330-500 μm) was used as an adsorbent. -1 An adsorption experiment similar to Example 1 was conducted. As a result, LDL was 704~/8, but after adsorption it remained at 667~/di (95%), and HDLch had 17η/di of 15~/di.
di (88chi), albumin is 3.517di is 3.
1r/dl (89qI)), which was not very selective.

Comparative example 3 CNB　r　Activated Sepharose 4B (Sweden).

D-glucosamitalonic acid (molecular weight: 193, having one carboxyl group) was immobilized on D-glucosamitalonic acid (molecular weight: 193, having one carboxyl group) by a conventional method. The ion exchange capacity of the obtained adsorbent was 21
It was μeq/-. When an adsorption experiment was conducted in the same manner as in Example 1, LDL was 704~/a was 67''9/dA!
(96%), HD L channel is 17m! ? /dl
is 16 mg/di (94%), albumin is 3.5
The gas/di was 3.5 f/dl (100%), meaning almost no adsorption was observed.

[Brief explanation of drawings]

The drawing is a sectional view showing an example of an adsorption device using the low-density riboprotein adsorbent of the present invention.

Claims (3)

[Claims] (1) It has a polyanion moiety with a molecular weight of 600 or more on the surface, and its negative charge density is 1 μeq /#// or more, 1 meq/m/! An adsorbent for adsorbing low-density riboproteins, which is characterized by: (2) The adsorbent for adsorbing low-density riboproteins according to claim 1, wherein the polyanion moiety having a molecular weight of 600 or more has a chain structure. (3) The adsorbent for adsorbing low-density riboproteins according to claim 1, wherein the polyanionic moiety having a molecular weight of 600 or more is a polyanionic moiety having at least one functional group exhibiting a negative charge per molecular weight of 300. .