JPS59206046A - Material for adsorbing lipoprotein of low specific weight - Google Patents

Abstract

PURPOSE:To adsorb selectively lipoprotein of low specific weight with high efficiency by using an adsorbent having many carboxylic groups in a molecule and polyanionic moiety having relatively large mol.wt. on its surface. CONSTITUTION:An adsorbent consisting of molecules having >=600mol.wt. and having many carboxylic groups in a molecule, such as polyacrylic acid, polymethacrylic acid, polyacrylic ester, polyacrylamide, etc., and having polyanionic moiety on its surface, is used to provide a catalyst capable of selectively adsorbing lipoprotein having low specific weight with high efficiency. Preferred mol.wt. of the polyanionic moiety is >=600, more pref. >=5,000.

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. 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 is expected to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even hasten the healing process. 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, al, : A new
approach to the management
to of family hypercholeg
terolemia, Removal of pl
asma-cholestyolbased on
the principle of affinit
ychromatography, Lancet, 2
1261-1264, 1976) and chromatography using glass powder or glass beads (Carlson, L.A.).

:Chromatographic 5eparati
on of serum 1ipoprotein
n glass powder columa, De
script of the method a
nd some applications, Cl
1n, Chim, Aeta.

5:52B-538, 1960. ).

However, although the adsorbent in which heparin is immobilized on agarose exhibits selective adsorption ability for low-density lipoproteins, the adsorption ability is insufficient, and since agarose is used as a carrier, the mechanical strength is limited to the non-photosensitive area. It is difficult to use, has poor operability, and can easily become clogged when body fluids are poured into it.
The bore was destroyed during the sterilization process, making it extremely 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 provide a method that can be widely used, selectively adsorbs low-density riboproteins with high efficiency, and non-selectively adsorbs low-density riboproteins. The purpose of the present invention is 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 have discovered that an adsorbent having a large number of carboxyl groups in its molecules and a polyanion moiety with a relatively large molecular weight on its surface has surprisingly high efficiency in producing low-density lipocarbons. The present inventors have discovered that it adsorbs proteins and has little non-selective adsorption of immunoglobulin, albumin, complement, fibrinogen, etc., and has completed the present invention.

That is, the present invention is a low-density lipoprotein adsorbent characterized by having a carboxyl group on the surface as a bamboo substituent that exhibits a negative charge and a polyanion portion having a molecular weight of at least 600. It is preferable that the polyanion moiety having a molecular weight of at least 600 has a chain structure and a carboxyl group in the side chain;
It is preferred that the polyanion moiety having a molecular weight of 0.00 has at least one carboxyl group per molecular weight of 300.

The target adsorbent of the present invention is low-density riboprotein, and to explain it in more detail, it has a molecular weight of 2.2X,
106 to 3.5 x 10', hydration density 1.005
to 1.034 (ff/ml), flotation coefficient (1,06
5) is 0 to 20 X 10-1sGIZ fist 5ec-
'・dyn-'・ff'' s diameter is 20.0 to 30
．． 0 nm lipoprotein (5CANU, A, M.

: plasma 1ipoproteins : a
n1ntroduction.

The Biochemistry of Ather
osclerosis”ed, by 5CANIJ A
, M., 1979, p. 3-B). This is a small lipoprotein with a buoyancy coefficient (1,065) of 20 x 10-'3 Brass, Sr'
Lipoproteins larger than 1 dyn-"-9-' may be adsorbed, but high-density lipoproteins with a high specific gravity are preferably 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 one molecule contains a carboxyl group (-COO
H, -COO-). To illustrate, polyacrylic acid: polymethacrylic acid; hand ψ49 koji →
Styrene-maleic acid 5-copolymer; hydrolyzate of polyacrylic acid ester, polyacrylic acid amide, polyacrylic acid nitrile, etc.: poly-
Synthetic products such as L-glutamic acid and polyaspartic acid, and acidic polysaccharides such as polygalacturonic acid and nialginic acid are used.

In addition, since the low-density lipoprotein, which is the substance to be adsorbed, is a huge lipoprotein with a diameter of about 200^, it is preferable that the structure of the polyanion part is a chain structure. is preferred. The density of carboxyl groups in the polyanion moiety is preferably at least one per molecular weight of 300. More preferably,
1 or more per 200 molecular weight, 1 per 70 molecular weight
It is desirable that there be one in every 50 units. The molecular weight referred to here includes the molecular weight of carboxyl groups.

The molecular weight of the polyanion moiety must be at least 600, since the smaller the molecular weight, the more it will adsorb low-density lipoproteins. Preferably it is 5000 or more, and 250
Nodes of 00 to 250,000 are desirable.

It is thought that the many carboxyl groups possessed by the 6-polyanion moiety bind the low-density lipoprotein with strong Coulomb force by recognizing multiple points on the low-density lipoprotein.

The method for producing the adsorbent of the present invention will be described below, taking as an example a method for producing an adsorbent for affinity chromatography, in which a carrier is activated and a ligand is bound thereto.

The press only needs to be able to immobilize a polyanion having a carboxyl group and a molecular weight of at least 600. Either a hydrophilic carrier or a hydrophobic carrier can be used, but when using a hydrophobic Iu carrier, albumin is sometimes added to the carrier. Hydrophilic carriers give more favorable results as non-specific adsorption occurs.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, inner-wall thread, or membrane, but the amount of retained polyanion having a carboxyl group and a molecular weight of at least 600, absorption 'f'J From the viewpoint of ease of handling as a U, particles or fibers are preferable.

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 particle size is defined as the particle size of each type, 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 2,500 μm or more, the adsorption amount and rate of low-density lipoprotein will decrease, and if it is less than 25 μm, activation of the coagulation system and blood cell adhesion will easily occur. Particulate carriers that can be used include agarose-based, dextran-based,
Examples include cellulose-based, polyacrylamide-based, glass-based, silica-based, activated carbon-based, etc., but hydrophilic carriers having a gel structure give good results. In addition, usually immobilized enzyme,
Known carriers used in affinity chromatography can be used without particular limitations. here,
It is necessary that the protein exclusion limit molecular weight of the carrier is 2 million or more, preferably 2.5 million to 10 million, and 30 million to 10 million.
Preferably, it is in the range of 00,000 to 7,000,000.

Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention have a carboxyl group on their surface, can immobilize a polyanion having a molecular weight of at least 6000, and have an average pore diameter in the range of 300 to 70 OA. For the polymer composition, known polymers that can have a porous structure, such as polyamides, polyesters, polyurethanes, and vinyl compound polymers, can be used, but in particular, porous vinyl compounds made hydrophilic with hydrophilic monomers can be used. Polymeric particles give favorable results.

Since the adsorption amount of the present invention is 11, it is used for purification treatment of body fluids, so it is necessary that no clogging occurs when body fluids are poured. 17'k Therefore, the phase used in the present invention is preferably a hard phase, and synthetic polymer carriers, inorganic carriers, etc. are preferably used.

The hard pressed body here refers to a carrier whose physical property values maintain a certain value or more when an external force is applied. Specifically, gel is filled into a container with a diameter of 10 m and 11 m and a length of 50 ff and 1 m. When water is passed through the column, it is preferable that the volume reduction of the gel is 10% or less when the difference between the inlet pressure and the outlet pressure of the column is 200++ll+Hg.

The porous structure has an average pore diameter of 20ON to 3000A.
However, if the average pore size is too small, the amount of low-density lipoprotein adsorbed will be small, and if it is too large, the strength of the polymer particles will decrease and the surface area will decrease. impractical as it decreases. The surface area is preferably at least 10 m2/f, preferably 55 m2/7
It is more preferable that it is above. Preferably 10
0 m27? That's all.

The total average pore diameter was measured using a mercury intrusion porosimeter. This method is a method in which mercury is injected into a porous material, and the pore volume is determined from the amount of mercury that has entered, and the pore diameter is determined from the pressure required for the intrusion. Pores with 40 or more holes can be measured. The pores of the present invention = 10 = are defined as communicating pores from the surface with a pore diameter of 4OA or more. The average pore diameter is the value of r when the value of dv/dlogr becomes the maximum, where V is the cumulative amount of pores measured using an r1 porosimeter.

When a fibrous carrier is used, it is preferable that the fiber diameter is 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. As the fibrous carrier that can be used, generally known grade fibers such as regenerated cellulose fibers, nylon, acrylic fibers, and polyester fibers can be used.

A method for immobilizing a polyanion having a carboxyl group and having a molecular weight of at least 600 on the surface of an insoluble carrier is as follows:
Any known method can be used, such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the polymer surface, but considering the elution properties of polyanions, it is recommended to use covalent bonds to identify and insolubilize the polyanion. is preferred.

Therefore, known methods of activating carriers and binding methods with ligands that are usually used in immobilized enzymes, affinity chromatography, and affinity chromatography can be used.

Examples of activation methods include halogenated cyanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, and 1,1'-carbonyldiimidazole method. 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 a 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 threads and glass.

Two or more types of polyanions having carboxyl groups and having a molecular weight of at least 600 may be bonded to the carrier. The amount of polyanion immobilized on the carrier is 10μ
f/me or more is required, from 10OtI/d to 40I
The range of n9/- is preferable, from 0.5η/- to 20η/
- is more preferable.

Above, the method for manufacturing the adsorbent of the present invention, in which a carrier is activated and then a polyanion having a carboxyl group and a molecular weight of at least 600 is bonded, has been described in detail. However, the present invention is not limited to this. isn't it.

For example, a method of monopolymerization (copolymerization) using a polymerizable monomer or crosslinking agent having a polyanion moiety having a carboxyl group and a molecular weight of at least 600; A method using a crosslinking agent having a polyanionic moiety having a molecular weight of at least 600 and a molecular weight of at least 600 can also be used. It is also possible to adopt a method in which a polyanion having a carboxyl group and a molecular weight of at least 6000 is activated and then bonded to the phase.

-16- That is, the present invention exhibits its effects by having a polyanion moiety having a carboxyl group and a molecular weight of at least 6oo on the surface of the adsorbent.
It doesn't depend on the manufacturing method.

The low-density lipoprotein 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 lipoprotein 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.
A cab 6 having a body fluid inlet is screwed into the cylinder 2, and 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' having a filter 3' on the inside. A container is formed, and an adsorbent is filled and held in the gap between the filters 3 and 3' to form the entire adsorbent layer 9.

The adsorbent layer 9 may be filled with the low-density lipoprotein adsorbent of the present invention alone, mixed with other adsorbents, or stacked. Other adsorbents that can be used include, for example, activated carbon, which has a wide range of adsorption capacities. By using this method, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. When used for extracorporeal circulation, the appropriate volume of the adsorbent layer 9 is about 50-400. 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 cavity-type plasma separator.The plasma components are passed through the device, purified, and then separated into blood cells. One method is to return the blood to the body together with its components, and the other method is to directly pass blood taken from the body through a nuclear device for purification.

In addition, regarding the passage rate of blood or plasma, since the adsorbent's adsorbent groups are very cylindrical, 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 adjusted. A high passing speed can be provided regardless of the situation. Therefore, a large amount of body fluid can be treated.

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

As described above, the adsorbent of the present invention selectively adsorbs and removes low-density lipoproteins in body fluids at a high rate, and the adsorption device using the adsorbent is extremely compact, simple, and safe. It is. In addition, it is less likely to non-selectively adsorb components that play an important role, such as immunoglobulin fibrinogen and complement in plasma proteins, and can adsorb low-density riboproteins with high efficiency, as well as coagulation fibrinolysis and complement system. is less likely to be activated.

INDUSTRIAL APPLICATION This invention is applicable to the general usage of purification and regeneration of body fluids, such as a lipemia, and is effective for the safe and reliable treatment of the disease caused by hyperlipidemia.

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

Example 1 A carrier with an average pore diameter of 400^ and a pore surface area of 9
0 m2/f silica gel 2.5? (10ml) was placed in a 10% by weight acetone solution of 3-aminopropyltriethoxysilane (62.5ml) and reacted for 40 hours under shaking at 50c. After this, the plate was washed with a light amount of acetone, then with distilled water, and replaced with phosphate buffer (pH 4.0). Next, polyacrylic acid (molecular weight 19,000
0-5400 0゜Molecular weight: 1 carboxyl group per 72) 200rn9.1-Cyclohexyl-6
-(2-morpholinoethyl)carbodiimide-meth-p
-) Suspended in 100 ml of phosphate buffer (pH 4,0) containing luenesulfonate 2000In9. After reacting with shaking at room temperature for 20 hours, the mixture was thoroughly washed with water to obtain a low-density riboprotein adsorbent.

(2) Quantification of polyacrylic acid is 6.3m9/ml
It was get.

In the adsorption experiment, 5 volumes of human plasma and 1 volume of adsorbent were mixed, and 57
'Q, incubated for 5 hours. After adsorption, low-density riboproteins were measured by a heparin-calcium precipitation method, and immunoglobulin G (IgG), immunoglobulin-17-A (IgA), and complement C5c were measured by a single radial immunodiffusion method.

As a result, the amount of low-density lipoprotein before adsorption was 550 m9/d.
Z% IgG is 1300 In9/dt, IgA is 19
0"97 dl % C5c was 85 m9/dt, whereas in the plasma after adsorption, low-density lipoproteins decreased to 20% of the plasma concentration before g & g, but IgG,
IgA and C3e were 90%, 79%, and 7, respectively.
It wasn't much lower than 6 inches.

Example 2 Polymethacrylic acid (molecular weight 5
An adsorbent was prepared in the same manner as in Example 1, except that an adsorbent (having one carboxyl group per molecular weight of 86) was used.

The fixed amount of polymethacrylic acid is 6.6 m97ml g
It was et.

As a result of the same experiment as in Example 1, in the plasma after adsorption, the concentration of low-density lipoprotein was 26% of the plasma concentration before adsorption, but the concentration of IgG SIgA and C5c was 85%, respectively.
The numbers did not fall significantly, at 82% and 76%.

-18- Example 6 A carrier with an average pore diameter of 400 A and a pore surface area of 9
0 m”/f silica gel 2.5 f (10+++l)
20 of 6-crisidoxypropyltrimethoxysilane
Weight% acetone solution 62.5ml! Place it inside and react for 40 hours while shaking at 50C! :Ta. Thereafter, the plate was washed with a light amount of acetone, then with distilled water, and replaced with 0.1M carbonate buffer (pH 9,8).

Next, this activated gel was mixed with 0.1 M carbonate buffer containing 200 m9 of L-glutamic acid pentamer (molecular weight 666, with one carboxyl group per molecular weight 110.5).
00 ml. The mixture was allowed to react at 50'C for 20 hours with shaking, and then allowed to stand at room temperature for one week. Thereafter, the material was washed with light and water to obtain a low-density lipoprotein adsorbent.

The fixed amount of L-glutamic acid pentamer is 6.91Q/meg
It was el.

An adsorption experiment similar to Experimental Example 1 was conducted using this adsorbent.

As a result of adsorption experiments, the concentration of low-density lipoprotein in plasma after adsorption was 42% of that before adsorption, but IgG,
IgA and C3c were 90%, 85%, and 8, respectively.
It wasn't much lower than 0chi.

Comparative Example 1 An activated gel obtained in the same manner as in Example 3 was treated with L-glutamic acid (molecular weight 147, having 2 carboxyl groups) 200
The suspension was suspended in 0.1M carbonate buffer 100ml containing m9, and the immobilization reaction was carried out in the same manner as in Example 3.

The fixed amount of L-glutamic acid is 7.9 m97ml ge
It was t.

Using the obtained adsorbent, an adsorption experiment was conducted in the same manner as in Example 3. As a result, in the plasma after adsorption, low-density lipoprotein was 85% of that before adsorption, and IgG, IgA, and C3c were:
No significant difference was observed at 90%, 85%, and 80%, respectively.

Example 4 Vinyl acetate 100f? , triallylisocyanurate) 4
1.4f (X=O040), ethyl acetate 1001, heptane 100f! , polyvinyl acetate (degree of polymerization 500)7.
51 and 2.2'-azobisisobutyronitrile 3,
82, water in which polyvinyl alcohol 1, 0.053T of sodium dihydrogen phosphate dihydrate and 1.5% by weight of disodium hydrogen phosphate dodecahydrate were dissolved 40 〇- was placed in a flask, thoroughly stirred, and then heated and stirred at 65C for 18 hours and then at 75C for 5 hours to carry out suspension polymerization to obtain a granular copolymer. After filtering and washing with water, then extracting with acetone, caustic soda 46.5
f and 2 t of methanol at 40 [18
For some time, the transesterification reaction of the copolymer was carried out.

The average particle size of the resulting gel was 1508 ml, and the vinyl alcohol unit (q OH) per unit weight was 9.0 meq.
/S', the specific surface area was 60 m''/?, and the exclusion limit molecular weight by dextran was 6 x 105.

Next, 10 g (dry weight) of the obtained gel was suspended in 120 g of dimethyl sulfoxide, and to this was added 8.5 rrt of epichlorohydrin, 2 g of 50% sodium hydroxide.
1-ram 10- was added and the activation reaction was carried out with stirring at 60C for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. Next, this activated gel was mixed with sodium polyacrylate (same as used in Example 1) 2. 1oo of 0.1M sodium carbonate buffer (pH 9,8) containing Or.

- suspended in The immobilization reaction was carried out at 50C for 20 hours with constant stirring, and then 60.6m97ml of Tris (
Hydroxyethyl) aminomethane solution g3s7 was added, and a blocking reaction (to block remaining active groups) was carried out for an additional 5,005 hours with stirring. Thereafter, it was thoroughly washed with water to obtain a low-density lipoprotein adsorbent. The amount of sodium polyacrylate fixed on this adsorbent was 7.4 mg.
/ ml ge L.

When the adsorbent was compared with the original gel particles and observed under an optical microscope, no damage such as chips or cracks was observed.

This adsorbent has an inner diameter of 101×11. Column 2 of length 50 dragons
Fill the bottles with 0.4 ml of ACD-added plasma in one bottle.
20ml of blood was added to the other tube at a flow rate of mil+, and 35ml of ACD-added blood was added to the other tube at a flow rate of 0.7-22-rnt/ml. There was no decrease in packing volume, clogging, or decrease in flow rate in any of the columns, and the pressure difference before and after the column was 10 mmHg or less for plasma, and was in the range of 10 to 20[++Hg] even for increased surface area.

When we examined the changes in plasma protein and blood cell components before and after passing through the column, we found that in plasma, the amount of low-density riboprotein after adsorption decreased to 26%, but 1g0% IgA.

C5c was no less impressive, with scores of 96%, 82%, and 78%, respectively.

1. When creating the column, red blood cells, white blood cells, before and after m.
III [No significant difference was observed in the degree of platelets.

[Brief explanation of drawings]

The drawing is a sectional view showing an example of an adsorption device using the low-density lipo can white matter adsorbent of the present invention. −23=

Claims (1)

[Scope of Claims] (11) A low-density lipoprotein adsorbent having a carboxyl group on the surface as a substituent that exhibits a negative charge and a polyanionic moiety having a molecular weight of at least 600. Claim 1, wherein the polyanion moiety having a molecular weight of at least 600 has a chain structure and has a carboxyl group in the side chain.
The low-density riboprotein adsorbent described in Section 1. (The low-density lipoprotein adsorbent according to claim 1, wherein the polyanion portion having 31 carboxyl groups and a molecular weight of at least 600 has at least one carboxyl group per molecular weight of 300.