JPS6259975B2 - - 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 an adsorbent in which heparin is immobilized on agarose gel (Lupien P-J, et.al.: A new approach
to the management of familial
hypercholesterolemia.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 very 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 surprisingly discovered an adsorbent that has a large number of negatively charged substituents in the molecule and a polymeric polyanion moiety with a large molecular weight as a ligand on the surface of an insoluble carrier. The present inventors have discovered that low-density lipoproteins can be adsorbed with extremely high efficiency, 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 polymer polyanion portion with a molecular weight of 25,000 or more as a ligand on the surface of an insoluble carrier, and the polymer polyanion portion with a molecular weight of 25,000 or more is Preferably, it is a polymeric polyanion moiety with a chain structure, and the molecular weight is
The polymeric polyanion moiety having a molecular weight of 25,000 or more contains at least 1 substituent having a negative charge per 1,000 molecular weight.
Preferably, it is a polymeric polyanion moiety having an individual structure.

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 riboprotein (SCANU, AM: plasma
lipoproteins：an introduction.
Biochemistry of Atherosclerosis”ed.by
SCANU AM, 1979, P.3-8). Lipoproteins with a lower specific gravity, i.e.
The levitation coefficient (1.063) is 20×10 ^-13・sec ^-1・dyn ^-1・
Lipoproteins larger than g ^-1 may be adsorbed, but high-density lipoproteins with a high specific gravity are preferably not adsorbed.

The polymer polyanion moiety referred to in the present invention is one whose molecular weight is 25,000 or more, and which contains carboxyl groups, sulfonic acid groups, phosphoric acid groups, etc. refers to a substance that has many substituents that exhibit negative charges. For example, acidic polysaccharides such as polygalaccuronic acid, phosphorylated mannan, chondroitin, chondroitin sulfate A, chondroitin C, hyaluronic acid, alginic acid,
Polyacrylic acid, polymethacrylic acid, styrene
Maleic acid copolymer, polystyrene sulfonic acid,
Examples include synthetic polymer polyanions such as polyethylene sulfonic acid, poly-α-methylstyrene sulfonic acid, polyvinyl phosphoric acid, polyphosphate ester, polyvinyl sulfonic acid, polystyrene phosphoric acid, and poly-L-glutamic acid.

In addition, since the low-density lipoprotein, which is the target substance for adsorption, is a huge lipoprotein with a diameter of approximately 200 Å, the structure of the polymeric polyanion part is preferably a chain structure, and it extends long from the surface of the adsorbent. It's better to be there. Furthermore, the density of substituents exhibiting negative charges in the polymeric polyanion moiety is preferably such that there is at least one substituent exhibiting negative charge per unit of molecular weight 1000, and more preferably one or more substituents exhibiting negative charge per unit of molecular weight 300. It is desirable that there be one per molecular weight unit of 70 to 200. The molecular weight referred to herein also includes the molecular weight of substituents that exhibit negative charges. Furthermore, among the functional groups exhibiting a negative charge, carboxyl groups exhibit particularly good adsorption performance. The molecular weight of the high-molecular polyanion moiety must be at least 25,000, since it will not adsorb low-density lipoproteins as much as it becomes small. It is preferably 30,000 or more, more preferably in the range of 50,000 to 250,000.

It is thought that the large number of negative charges possessed by the polymeric polyanion moiety recognizes the large number of points on the low-density lipoprotein, thereby binding the low-density lipoprotein with a strong Coulomb 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 only needs to be able to immobilize a polymeric polyanion with a molecular weight of 25,000 or more, and both hydrophilic and hydrophobic carriers can be used; however, when using a hydrophobic carrier, nonspecific adsorption of albumin to the carrier sometimes occurs. Therefore, hydrophilic carriers give more favorable results.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but from the viewpoint of the amount of polymer polyanion with a molecular weight of 25,000 or more retained and ease of handling as an adsorbent, particles condition,
Fibrous ones are preferred.

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. When the average particle size is 2500 μm or more, the adsorption amount and adsorption rate of low-density lipoprotein decreases, and when 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, activated carbon, 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 2.5 million to 10 million, and 300 million to 10 million.
More 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 a molecular weight of 25,000 on their surface.
The average pore diameter is in the range of 200 Å to 3000 Å, more preferably in the range of 250 Å to 1000 Å, and desirably in the range of 300 to 700 Å. 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 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 mentioned here refers to one whose physical properties maintain a certain value or more when an external force is applied to it.
When filling a container and passing water through the column, it is preferable that the volume reduction rate of the gel is 10% or less when the difference between the inlet pressure and outlet pressure of the column 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. where the surface area is at least 10 m ² /g
It is preferably at least 55 m ² /g, more preferably at least 55 m 2 /g. 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 infiltrated, and the pore diameter is determined from the pressure required for intrusion, and pores of 40 Å or more can be measured. What is the hole of the present invention?
Defined as communicating pores 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 becomes the 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 polymeric polyanions on the surface of insoluble carriers 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 high molecular weight polyanion, it is preferable to use it after fixation and insolubilization by covalent bonding. Therefore, it is possible to use commonly known methods for activating immobilized enzymes, carriers used in affinity chromatography, and binding methods for ligands.

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.

There is no problem even if two or more types of polymeric polyanions referred to in the present invention are bonded to the carrier. The amount of the polymeric polyanion immobilized on the carrier needs to be 10 μg/ml or more, preferably in the range of 100 μg/ml to 40 mg/ml, and more preferably in the range of 0.5 mg/ml to 20 mg/ml.

As for the method for manufacturing the adsorbent of the present invention, the method of activating the carrier and then bonding the polymeric polyanion has been described above in detail, but the present invention is not limited thereto.

For example, in the present invention, a method of polymerizing (copolymerizing) using a polymerizable monomer or a crosslinking agent having a polymeric polyanion portion, or adding a crosslinking agent having a polymeric polyanion portion to the crosslinked polymer particles at the time of post-crosslinking. It is also possible to adopt a method using the above method. Alternatively, a method of activating a polymeric polyanion and then binding it to a carrier can also be adopted.

That is, the present invention exhibits its effects by having a polymeric polyanion portion 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 opening of the cylinder 2 through a packing 4 with a filter 3' stretched thereon. 8 are screw-fitted 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 wide range of clinical effects can be expected due to the synergistic effect of the adsorbent. The volume of the adsorbent layer 9 is 50 to 400 when used for extracorporeal circulation.
ml is appropriate. When the device of the present invention is used for extracorporeal circulation, there are roughly two methods as follows.
One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane 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 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 an adsorption device using the adsorbent is extremely compact, simple, and safe. It is. and,
immunoglobulin fibrinogen in plasma proteins,
It rarely non-selectively adsorbs components that play an important role such as complement, can adsorb low-density lipoproteins with high efficiency, and is less likely to cause coagulation fibrinolysis or activate the complement system.

The present invention is applicable 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.

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

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 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).

This activated gel was then suspended in 100 ml of 0.1 M carbonate buffer containing 200 mg of sodium alginate (molecular weight 32,000-240,000). The reaction was carried out 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 sodium alginate is 6.7mg/mlge
It was hot.

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 amount of low-density lipoprotein before adsorption was 400
mg/dl, IgG 1100mg/dl, IgA 250mg/dl,
C3c was 90 mg/dl, whereas low-density lipoprotein was 19 mg/dl in the plasma after adsorption.
%, but IgG, IgA, and C3c were each 92%.
%, 83%, and 78%, which were not very low.

Comparative Example 1 An activated gel obtained in the same manner as in Example 1 containing 200 mg of heparin sodium (molecular weight 7000-20000)
Example 1
The immobilization reaction was carried out in the same manner as described above. The fixed amount of heparin sodium was 7.1 mg/ml gel.

Using the obtained adsorbent, an adsorption experiment was conducted in the same manner as in Example 1. As a result, the amount of low-density lipoprotein in plasma after adsorption was only 70% of that before adsorption.
IgG, IgA, and C3c were 90%, 89%, and 76%, respectively.
It was hot.

Example 2 An activated gel obtained in the same manner as in Example 1 was suspended in 100 ml of 0.1M carbonate buffer containing 200 mg of polygalacturonic acid (molecular weight 60,000 to 80,000), and an immobilization reaction was performed in the same manner as in Example 1. Ta.

The fixed amount of sodium polygalacturonate is 6.8
mg/mlgel.

The obtained adsorbent was left as a low-density lipoprotein adsorbent, and an adsorption experiment was conducted in the same manner as in Example 1. As a result, the amount of low-density lipoprotein in plasma after adsorption decreased to 25% of that before adsorption, whereas IgG,
IgA and C3c did not fall much, at 90%, 85%, and 80%, respectively.

Example 3 Activated gel obtained in the same manner as in Example 1 was treated with sodium hyaluronate (molecular weight 100000-10000000)
It was suspended in 100 ml of 0.1M carbonate buffer containing 200 mg, and the immobilization reaction was carried out in the same manner as in Example 1.

The fixed amount of sodium hyaluronate is 5.4mg/ml
It was hot with gel.

The obtained adsorbent was used as a low-density lipoprotein adsorbent, and an adsorption experiment was conducted in the same manner as in Example 1. As a result, the amount of low-density lipoprotein in plasma after adsorption decreased to 25% of that before adsorption, whereas IgG,
IgA and C3c did not fall much, at 85%, 82%, and 76%, respectively.

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
Pour 400ml of water containing 1.5% by weight into a flask, stir well, and heat at 65°C for 18 hours.
Suspension polymerization was carried out by heating and stirring at °C 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 (q OH) 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 alginate. The immobilization reaction was carried out at 50°C for 20 hours with stirring, then 33 ml of a 60.6 mg/ml tris(hydroxyethyl) aminomethane solution was added, and further
A blocking reaction (to block remaining active groups) was carried out at 50°C for 5 hours with stirring. Thereafter, it was thoroughly washed with water to obtain a low-density lipoprotein adsorbent. The amount of sodium alginate fixed on this adsorbent is
It was 8.9 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 crumbling 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 10 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, the amount of low-density lipoproteins after adsorption decreased to 21%, but IgG, IgA,
C3c was not much lower at 93%, 85%, and 77%, 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)

[Claims] 1. A ligand with a molecular weight on the surface of an insoluble carrier.
A low-density lipoprotein adsorbent characterized by having a polymeric polyanine moiety having a molecular weight of 25,000 or more. 2. The low-density lipoprotein adsorbent according to claim 1, wherein the polymeric polyanion portion having a molecular weight of 25,000 or more is a polymeric polyanion portion with a chain structure. 3. The low-density lipoprotein adsorbent according to claim 1, wherein the polymeric polyanion portion having a molecular weight of 25,000 or more is a polymeric polyanion portion having at least one substituent that exhibits a negative charge per molecular weight of 1,000. .