JPS6259976B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a low-density lipoprotein adsorbent that selectively adsorbs and removes low-density lipoproteins, 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 lipoproteins, is thought to be closely related to the cause or progression of arteriosclerosis. As arteriosclerosis progresses, the possibility of developing serious circulatory system symptoms such as myocardial infarction and cerebral infarction becomes 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 that it would prevent the progression of the above-mentioned diseases, alleviate their symptoms, and even hasten their healing.

Existing techniques that can be used for the above purpose include adsorption using adsorbents in which heparin is immobilized on agarose gel (Lupien, P-J, et.al.: A new
approach to the management of familial
hypercholeste−rolemia.Removal of plasma−
cholesterol based on the principle of affinity
chromatography.Lancet, 2:1261-1264,
1976.) and chromatography using glass powder or beads (Carlson, L.
A.：Chromatographic separation of serum
lipoproteins on glass powder columns.
Description of the method and some
applications.Clin.Chim.Acta, 5:528-538;
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 was difficult to use because it had poor performance and operability, was prone to clogging when body fluids were poured into it, and the pores were destroyed during sterilization.

Furthermore, methods using glass powder or glass beads have the drawbacks of low adsorption capacity and low adsorption selectivity, and are not practical.

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 lipoproteins 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 have discovered that an adsorbent having a large number of carboxyl groups in the molecule and a polyanion moiety with a relatively large molecular weight as a ligand on the surface of an insoluble carrier has surprisingly high efficiency. The present inventors have discovered that low-density lipoproteins can be adsorbed using the method, and non-selective adsorption of immunoglobulin, albumin, complement, fibrinogen, etc. is small, and the present invention has been completed.

That is, the present invention is a low-density lipoprotein adsorbent characterized by having a carboxyl group as a ligand and a substituent showing a negative charge on the surface of an insoluble carrier, and a polyanionic moiety having a molecular weight of at least 600. a polyanionic moiety having a molecular weight of at least 600,
It is preferable that it has a chain structure and has a carboxyl group in its side chain, and the polyanion part that has a carboxyl group and has a molecular weight of at least 600 has at least one carboxyl group per molecular weight of 300. It is preferable that there be.

The target adsorbent of the present invention is low-density lipoprotein.
2.2×10 ⁶ to 3.5×10 ⁶ , hydrated density from 1.003
1.034 (g/ml), levitation coefficient (1.063) from 0 to 20
×10 ^-13 cm・sec ^-1・dyn ^-1・g ^-1 , diameter from 20.0
30.0nm lipoprotein (SCANU, AM: plasma
lipoproteins：an introduction.
Biochemistry of Atherosclerosis”ed.by
SCANU AM, 1979, P.3-8). Lipoproteins with a specific gravity smaller than this, that is, the buoyancy coefficient (1.063) is 20×10 ^-13 cm・sec ^-1・
Lipoproteins larger than dyn ^-1 ·g ^-1 may be adsorbed, but high-density lipoproteins with a high specific gravity are preferably not adsorbed.

The polyanion moiety used in the present invention refers to a polyanion moiety having a molecular weight of 600 or more and a large number of carboxyl groups (-COOH, -COO ^- ) in one molecule. Examples include polyacrylic acid; polymethacrylic acid; styrene-maleic acid copolymer; hydrolysates of polyacrylic acid ester, polyacrylic acid amide, polyacrylic acid nitrile, etc.; poly-L-glutamic acid, polyaspartic acid, etc. Examples include synthetic compounds and polygalacturonic acid; acidic polysaccharides such as alginic acid.

In addition, since low-density lipoproteins, which are the target substances for adsorption, are huge lipoproteins with a diameter of approximately 200 Å, it is preferable that the structure of the polyanion moiety is a chain structure, and it is better to extend long from the surface of the adsorbent. preferable. Further, the density of carboxyl groups in the polyanion moiety is preferably at least one per 300 molecular weight. More preferably, the molecular weight
The number is one or more per 200, and preferably one per unit of molecular weight 70 to 150. The molecular weight referred to here includes the molecular weight of carboxyl groups. The molecular weight of the polyanion moiety needs to be at least 600, since it will not adsorb low-density lipoproteins as much as it becomes small. Preferably it is 5000 or more,
A range of 25000 to 250000 is desirable.

It is thought that the many carboxyl groups of the polyanion moiety recognize multiple points on the low-density lipoprotein, thereby binding the low-density lipoprotein with a strong Coulombic force.

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 carrier has a carboxyl group and at least
It is sufficient to immobilize a polyanion with a molecular weight of 600, and 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 it is preferable to use a hydrophilic carrier. gives a more favorable result.

The shape of the insoluble carrier may be any known shape, such as particulate, fibrous, hollow fiber, or membrane, but the amount of polyanion that has a carboxyl group and a molecular weight of at least 600 and its handling as an adsorbent are important. Particulate and fibrous materials are preferable.

As a particulate carrier, the average particle size is 25 μm or less.
It is preferably in the range of 2500 μm. The average particle size is determined by classifying in running water using a sieve specified in JIS-Z-8801, and then taking the intermediate value between the upper limit particle size and lower limit particle size of each class as the particle size of each class, and calculating the average as the weight average. Calculate particle size. 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 lipoprotein decreases, and if the average particle size is 25 μm or less,
It is easy to activate the coagulation system and cause blood cell adhesion. Particulate carriers that can be used include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, silica-based and activated carbon-based carriers, 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 2.5 million to 10 million, and 300 million to 10 million.
Preferably, it is in the range of 10,000 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 carboxyl groups on their surfaces, can immobilize polyanions having a molecular weight of at least 600, and have an average pore diameter of 200.
Å to 3000Å, more preferably 250Å to
It is in the range of 1000 Å, preferably in the range of 300 to 700 Å. The polymer composition is polyamide,
Known polymers that can have a porous structure, such as polyester, polyurethane, and vinyl compound polymers, can be used, but particularly, vinyl compound porous polymer particles made hydrophilic with a hydrophilic monomer give preferable results. give.

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,
Synthetic polymer carriers, inorganic carriers, etc. are preferably used.

The hard carrier mentioned here means that when an external force is applied,
This refers to a carrier whose physical property values are above a certain value. Specifically, when gel is packed into a container with a diameter of 10 mm and a length of 50 mm, and water is passed through it, the inlet pressure and outlet pressure of the column are It is preferable that the volume reduction rate of the gel is 10% or less when the difference is 200 mmHg.

The porous structure has an average pore diameter of 200 Å to 3000 Å.
If the average pore size is too small, the amount of low-density lipoprotein adsorbed will be small; if it is too large, the strength of the polymer particles will decrease and the surface area will decrease. Therefore, it is not practical. The surface area is preferably at least 10 m ² /g, more preferably 55 m ² /g or more. It is desirably 100 m ² /g 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 volume is determined from the amount of mercury that has entered, and the pore diameter is determined from the pressure required for intrusion. Pores larger than 40 Å can be measured. A pore in the present invention is defined as a pore communicating from the surface with a pore diameter of 40 Å or more. The average pore diameter is the value of r when the value of dv/dlogr is maximum, where r is the pore diameter and V is the cumulative pore volume measured with a porosimeter.

When using a fibrous carrier, the fiber diameter is 1
μm to 50 μm, more preferably 5 μm to 25 μm
Preferably it is in the μm range. 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 fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used.

Methods for immobilizing polyanions having a carboxyl group and a molecular weight of at least 600 on the surface of an insoluble support include covalent bonding, ionic bonding, physical adsorption,
Any known method such as embedding or insolubilization by precipitation on the surface of the polymer can be used, but considering the elution properties of the polyanion, it is preferable to fix and insolubilize the polyanion by covalent bonding. For this purpose, known methods for activating carriers and methods for binding to ligands that are usually used in immobilized enzymes, affinity chromatography, and affinity chromatography can be used.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. 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.

Furthermore, various silane coupling agents are preferably used for silica-based, glass-based, and other silanol group-containing carriers.

The carrier has a carboxyl group and at least
It is possible to combine two or more types of polyanions with a molecular weight of 600. The amount of polyanion immobilized on the carrier is required to be 10 μg/ml or more, and 100 μg/ml or more is required.
A range of μg/ml to 40mg/ml is preferred, and 0.5
More preferably, the range is from mg/ml to 20 mg/ml.

As described above, as a method for producing the adsorbent of the present invention, after activating the carrier, the carrier has a carboxyl group and at least
Although the method of bonding a polyanion having a molecular weight of 600 has been described in detail, the present invention is not limited thereto.

For example, it has a carboxyl group and at least
A method of polymerization (copolymerization) using a polymerizable monomer or crosslinking agent having a polyanion moiety with a molecular weight of 600, and a polyanion having a carboxyl group and a molecular weight of at least 600 at the time of further post-crosslinking into crosslinked polymer particles. A method using a crosslinking agent having 10% or more 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 600 is activated and then bonded to a carrier.

That is, the present invention exhibits its effects by having a polyanion moiety having a carboxyl group and a molecular weight of at least 600 on the surface of the adsorbent, and is not dependent 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 filter 3 is installed inside an opening at one end of a cylinder 2.
A cap 6 having a body fluid inlet is screwed into the cap 6 through a packing 4 which has a body fluid inlet, and a cap having a body fluid outlet 7 through a packing 4' which has a filter 3' stretched inside the opening at the other end of the cylinder 2. 8 are screwed together to form a container, and an adsorbent layer 9 is formed by filling and holding an adsorbent in the gap between the filters 3 and 3'.

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. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. When used for extracorporeal circulation, the volume of the adsorbent layer 9 is 50~
Approximately 400ml is appropriate. When using the device of the present invention for extracorporeal circulation, there are roughly two methods as follows. First, blood taken from the body is separated into plasma components and blood cell components using a centrifuge or a membrane plasma separator, and the plasma components are passed through the device to be 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 the device for purification.

In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely high, 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. Regardless, high passing speeds can be provided. 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. It is less likely to non-selectively adsorb components that play important roles, such as immunoglobulin fibrinogen and complement in plasma proteins, and can adsorb low-density lipoproteins with high efficiency, as well as coagulation and fibrinolysis. It is less likely to activate the complement system.

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

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

Example 1 As a carrier, 2.5 g (10 ml) of silica gel with an average pore diameter of 400 Å and a pore surface area of 90 m ² /g was
The mixture was poured into 62.5 ml of a 10% by weight acetone solution of aminopropyltriethoxysilane, and reacted at 50°C with shaking for 40 hours. After this, wash with a sufficient amount of acetone, distilled water, and phosphate buffer (PH).
4.0). Next, phosphoric acid containing 200 mg of polyacrylic acid (molecular weight 190000 to 540000, with one carboxyl group per molecular weight 72) and 2000 mg of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-toluenesulfonate was added. Suspended in 100 ml of buffer (PH4.0). After reacting at room temperature for 20 hours with shaking, the mixture was thoroughly washed with water to obtain a low-density lipoprotein adsorbent.

The fixed amount of polyacrylic acid was 6.3 mg/mlgel.

In the adsorption experiment, 5 volumes of human plasma and 1 volume of adsorbent were mixed and incubated at 37°C for 3 hours. After adsorption, low-density lipoproteins were measured by a heparin-calcium precipitation method, and immunoglobulin G (IgG), immunoglobulin A (IgA), and complement C3c were measured by a single radial immunodiffusion method.

As a result, the low-density lipoprotein before adsorption was 350
mg/dl, IgG 1300mg/dl, IgA 190mg/dl,
C3c was 85 mg/dl, whereas low-density lipoprotein in the plasma after adsorption was 20 mg/dl below the plasma concentration before adsorption.
%, but IgG, IgA, and C3c were each 90%.
%, 79%, and 73%, which were not much lower.

Example 2 An adsorbent was prepared in the same manner as in Example 1, except that polymethacrylic acid (molecular weight 5000 to 50000, with one carboxyl group per molecular weight 86) was used instead of polyacrylic acid.

The fixed amount of polymethacrylic acid was 6.6 mg/ml gel.

As a result of the same experiment as in Example 1, it was found that in the plasma after adsorption, the concentration of low-density lipoprotein was 26% lower than the plasma concentration before adsorption.
%, but IgG, IgA, and C3c were each 85%.
%, 82%, and 76%, which were not very low.

Example 3 As a carrier, 2.5 g (10 ml) of silica gel with an average pore diameter of 400 Å and a pore surface area of 90 m ² /g was
The mixture was placed in 62.5 ml of a 20% by weight acetone solution of glycidoxypropyltrimethoxysilane, and reacted at 50°C with shaking for 40 hours. Thereafter, the solution was washed with a sufficient amount of acetone, then with distilled water, and replaced with 0.1M carbonate buffer (PH9.8).

Next, this activated gel was
The suspension was suspended in 100 ml of 0.1M carbonic acid buffer containing 200 mg of the polymer (molecular weight 663, with one carboxyl group per molecular weight 110.5). The mixture was reacted at 50°C for 20 hours with shaking, and then left at room temperature for one week. Thereafter, it was thoroughly washed with water to obtain a low-density lipoprotein adsorbent.

The fixed amount of L-glutamic acid pentamer is 6.9mg/ml
It was hot with gel.

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 are 90%, 85%, and 80%, respectively.
It wasn't too bad.

Comparative Example 1 An activated gel obtained in the same manner as in Example 3 was suspended in 100 ml of 0.1M carbonate buffer containing 200 mg of L-glutamic acid (molecular weight 147, has two carboxyl groups), and fixed in the same manner as in Example 3. A chemical reaction was carried out.

The fixed amount of L-glutamic acid was 7.9 mg/ml gel.

Using the obtained adsorbent, an adsorption experiment was conducted in the same manner as in Example 3. After adsorption, low-density lipoproteins in plasma were 85% of those before adsorption, IgG, IgA, and C3c.
were 90%, 85%, and 80%, respectively, with no significant difference.

Example 4 100 g of vinyl acetate, 41.4 g of triallylisocyanurate (X = 0.40), 100 g of ethyl acetate, 100 g of heptane, 7.5 g of polyvinyl acetate (degree of polymerization 500) and 3.8 g of 2,2'-azobisisobutyronitrile.
A homogeneous mixed liquid consisting of polyvinyl alcohol 1
wt%, sodium dihydrogen phosphate dihydrate 0.05 wt% and disodium hydrogen phosphate dodecahydrate
Add 400 ml of water in which 1.5% by weight was dissolved into a flask, stir thoroughly, and then heat at 65°C for 18 hours and then at 75°C.
Suspension polymerization was carried out by heating and stirring for 5 hours to obtain a granular copolymer. After washing with water and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.5 g of caustic soda and 2 methanol at 40° C. for 18 hours.

The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit (qOH) per unit weight was
The molecular weight was 9.0 meq/g, the specific surface area was 60 m ² /g, and the exclusion limit molecular weight by dextran was 6×10 ⁵ .

Next, 10 g (dry weight) of the obtained gel was suspended in 120 ml of dimethyl sulfoxide, and this was mixed with 78.3 ml of epichlorohydrin and 10 ml of 30% sodium hydroxide.
was added, and the activation reaction was carried out while stirring at 30°C for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. This activated gel was then suspended in 1000 ml of 0.1 M sodium carbonate buffer (PH 9.8) containing 2.0 g of sodium polyacrylate (same as used in Example 1).
The immobilization reaction was carried out at 50°C for 20 hours with stirring, then 33ml of a 60.6 mg/ml tris(hydroxyethyl)aminomethane solution was added, and the mixture was further heated at 50°C for 5 hours.
A blocking reaction (to block remaining active groups) was carried out with stirring for a certain period of time. Thereafter, it was thoroughly washed with water to obtain a low-density lipoprotein adsorbent. The amount of sodium polyacrylate fixed on this adsorbent is
It was 7.4 mg/ml gel.

When the adsorbent was compared with the original gel particles and observed under an optical microscope, no breakage such as chipping or flaking was observed.

This adsorbent was packed into two columns with an inner diameter of 10 mm and a length of 50 mm, one containing 20 ml of ACD-added plasma at a flow rate of 0.4 ml/min, and the other containing ACD-added blood at a flow rate of 0.7 ml/min.
35 ml was flowed at a flow rate of min. No decrease in packing volume, clogging, or decrease in flow rate was observed in any of the columns, and the pressure difference before and after the column was less than 100 mmHg for plasma and in the range of 10 to 20 mmHg for blood.

When we investigated changes in plasma proteins and blood cell components before and after passing through the column, we found that in plasma, low density lipoproteins after adsorption decreased to 23%, but IgG, IgA, and C3c
were not much lower at 93%, 82%, and 78%, respectively.

Furthermore, in blood, no significant difference was observed in the concentrations of red blood cells, white blood cells, and platelets before and after passing through the column.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. A low-density lipoprotein adsorbent characterized by having a polyanion moiety having a carboxyl group as a ligand and a substituent showing a negative charge and having a molecular weight of at least 600 on the surface of an insoluble carrier. 2. A polyanion moiety having a carboxyl group and a molecular weight of at least 600 has a chain structure,
The low-density lipoprotein adsorbent according to claim 1, which has a carboxyl group in its side chain. 3. The low-density lipoprotein adsorbent according to claim 1, wherein the polyanion portion having a carboxyl group and having a molecular weight of at least 600 has at least one carboxyl group per molecular weight of 300.