JPH043988B2 - - 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 diseases mentioned above, alleviate the symptoms, and even speed up the healing process.

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
hypercholesternle mia.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
Lipoprotein 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 and it is difficult to handle. 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 the molecule has a large number of functional groups exhibiting negative charges.
An adsorbent that has a polyanion moiety with a relatively large molecular weight on its surface and a negative charge density within a specific range adsorbs low-density lipoproteins with surprisingly high efficiency, and has little non-selective adsorption. The present inventors have also discovered that they are less likely to activate blood coagulation, fibrinolytic system, and complement system, leading to the completion of the present invention.

That is, the present invention provides a method for adsorbing low-density lipoproteins, which has a polyanion moiety with a molecular weight of 600 or more on the surface and has a negative charge density of 1 μeq/ml or more and 1 meq/ml or less. It is preferable that the polyanion part which is an adsorbent and has a molecular weight of 600 or more has a chain structure, and the polyanion part which has a molecular weight of 600 or more has a functional group that exhibits at least one negative charge per molecular weight of 300. A polyanion moiety having a group is preferable.

The target adsorbent of the present invention is low-density lipoprotein.
2.2×10 ⁶ to 3.5×10 ⁶ , hydrated density 1.003 to 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 to 30.0nm
lipoprotein (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 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 referred to in the present invention is a functional group having a molecular weight of 600 or more and having a negative charge in one molecule, that is, a sulfonic acid group (SO ₃ H,
refers to substances that have many functional groups that exhibit negative charges in plasma, such as SO ₃ ^- ). Examples include vinyl-based synthetic polyanions such as polyvinyl sulfonic acid and polyvinyl phosphoric acid, styrene-based polyanions such as polystyrene sulfonic acid and polystyrene phosphate, peptide-based polyanions such as polytaurine, RNA, and DNA.
Examples include nucleic acid polyanions such as polyphosphoric acid, polyphosphate ester, poly-α-methylstyrene sulfonic acid, and the like.

Among them, polyanions obtained by synthesis have excellent chemical stability, are stable against high-pressure steam sterilization, γ-ray sterilization, ethylene oxide sterilization, etc., and are easy to obtain, and molecular weight control is also relatively easy. Superior to natural products in terms of ease of use, etc.
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, those that can be graft-polymerized directly onto the carrier, such as vinyl anions, give more favorable results in that a polyanion having a large molecular weight can be fixed to the carrier in a high retention amount.

In addition, since the low-density lipoprotein, which is the target substance for adsorption, is a huge lipoprotein with a diameter of about 200 Å, it is preferable that the structure of the polyanion part is a chain structure, and it is better to extend it long from the surface of the adsorbent. preferable. Further, it is preferable that the polyanion moiety has at least one negative charge density per 300 molecular weight. More preferably, the number is one or more per molecular weight of 200, and desirably one per unit of molecular weight of 70 to 150. The molecular weight mentioned here is
It also includes the molecular weight of functional groups that exhibit negative charges. 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 25,000 to 1,000,000 is desirable.

It is thought that the polyanion moiety's many negatively charged functional groups recognize multiple points on the low-density lipoprotein, thereby binding the low-density lipoprotein with strong Coulomb force.

The density of negative charge is from 1 μeq per ml of adsorbent.
A range of 1 meq is an appropriate range that has good adsorption performance for low-density lipoproteins, good adsorption selectivity, and little influence on the coagulation fibrinolytic system and the complement system. When the negative charge density is lower than 1 μeq/ml, the adsorption capacity of low-density lipoproteins falls short of practical performance, and when it exceeds 1 meq, non-selective adsorption increases, adversely affecting the coagulation fibrinolytic system and the complement system. A more preferable range is from 5μeq/ml
700μeq/ml, more preferably from 10μeq/ml
From 500μeq/ml, more preferably from 20μeq/ml
It is 300μeq/ml.

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 producing the adsorbent of the present invention include, for example, activating a carrier and covalently bonding a chain synthetic polyanion at one end thereof, and graft polymerizing an anion monomer onto the carrier to form a polyanion graft chain. Can be mentioned.

The carrier only needs to be able to immobilize a polyanion with a molecular weight of at least 600 at a negative charge density in the range of 1 μeq/ml to 1 meq/ml, and both hydrophilic and hydrophobic carriers can be used, but when using a hydrophobic carrier, Since non-specific adsorption of albumin to the carrier sometimes occurs, a hydrophilic carrier gives 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 polyanion retained with a molecular weight of at least 600 and the ease of handling as an adsorbent, particulate is preferred. ,
Fibrous ones are preferred.

As a particulate carrier, the average particle size is 25μm or more.
It is preferably in the range of 2500 μm. The average particle size is determined by classifying in running water using a sieve as specified in JIS-Z-8801, then taking the intermediate value between the upper limit particle size and the adjusted particle size for each grade as the particle size, and calculating 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 less than 25 μm,
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-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 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. This means that the average pore diameter is in the range of 200 Å to 3000 Å, more preferably in the range of 250 Å to 700 Å, and desirably in the range of 300 to 650 Å.

Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymeric particles used in the present invention contain polyanions with a molecular weight of at least 600 at a negative charge density of 1 μeq/ml.
It can be immobilized in the range of 1meq/ml, and the polymer composition can be any known polymer that can have a porous structure, such as polyamide, polyester, polyurethane, or vinyl compound polymer. In particular, vinyl compound-based porous polymer particles made hydrophilic with a hydrophilic monomer give preferable 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,
Synthetic polymer carriers, inorganic carriers, etc. are preferably used.

The hard carrier mentioned here means that when an external force is applied,
It refers to a carrier whose physical property values are above a certain value. Specifically, when the 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 difference between the inlet pressure of the column and the outlet pressure It is preferable that the volume reduction rate of the gel is 10% or less when the temperature 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. 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 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
1μm to 50μm, more preferably 5μm to 25μm
It is good to have something within this 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 with a molecular weight of at least 600 on the surface of an insoluble support include covalent bonding,
Any known method such as ionic bonding, physical adsorption, embedding, or precipitation insolubilization 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. Therefore, it is possible to use an immobilized enzyme, a known method for activating a carrier used in affinity chromatography, a method for binding to a ligand, and a graft polymerization method using a carrier as a backbone polymer and a polyanion as a branch.

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 only needs to be able to perform a substitution and/or addition reaction with a nucleophilic reactive group having an active hydrogen such as an amino group, a hydroxyl group, or a thiol group of a ligand, and is not limited to the above-mentioned examples.
Considering chemical stability, thermal stability, etc., a method using 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.

Examples of graft polymerization methods include chain transfer method, emulsion polymerization method, graft polymerization method using various initiators such as cerium salt, persulfate-lithium halide, hydrogen peroxide-metal salt, perester group, mercapto group, etc. Examples include graft polymerization using a polymer having a functional group such as a diazo group, graft polymerization using air or ozone oxidation, and radiation grafting. A method of graft polymerizing an anionic monomer using a cerium salt, iron salt, etc. 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 to the inside of 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 producing the adsorbent of the present invention, and the polyanion having a molecular weight of at least 600 is
Although a method for binding to a carrier with a negative charge density of 1 μeq/ml to 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 600, a method of activating a polyanion having a molecular weight of at least 600 and then bonding it to a carrier, etc. can also be adopted. .

That is, in the present invention, a polyanion moiety having a molecular weight of at least 600 is added to the surface of the adsorbent at 1 μeq/ml.
By having a negative charge density of 1meq/ml from
This effect is exhibited 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 fitted with screws to form a container, and an adsorbent is filled and held between the filters 3 and 3' to form an adsorbent layer 9.

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. The volume of the adsorbent layer 9 is 50 to 40 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 the 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 lipoproteins can be adsorbed with high efficiency, and coagulation fibrinolysis and complement system activation are less likely to occur.

The present invention is applicable to the general use of purifying and regenerating carriers for hyperlipidemia, etc., 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 Graft polymerization of vinyl sulfonic acid was carried out using Sepharose 4B (manufactured by Pharmacia, Sweden) as a carrier. First, 10 ml (wet volume) of Sepharose 4B is immersed in 200 ml of distilled water, the air is replaced with nitrogen, then 35 mmol of vinyl sulfonic acid (having one sulfonic acid group per molecular weight of 108) is added, and finally 0.1 N nitric acid is added. 2.5 mmol of ceric ammonium nitrate was dissolved in 50 ml and added. Thereafter, graft polymerization was carried out under a nitrogen atmosphere at 35° C. for 3 hours with stirring. After the reaction, the mixture was thoroughly washed with water to obtain an adsorbent for adsorbing low-density lipoproteins. The ion exchange capacity, ie, negative charge density, of the obtained adsorbent was measured by a conventional method and found to be 52 μeq/ml.

In the adsorption experiment, 10 volumes of hyperlipidemic patient plasma and 1 adsorbent were used.
The contents were mixed and incubated at 37°C for 3 hours with shaking. After adsorption, low-density lipoproteins (hereinafter abbreviated as LDL) were extracted by the heparin-calcium precipitation method, high-density lipoprotein cholesterol (hereinafter abbreviated as HDLch) by the heparin-manganese precipitation method, and albumin was extracted by the bromcresol green method. Measured at The results of the analysis show that LDL in plasma
was 704 mg/dl, while after adsorption it was 310 mg/dl.
dl (44% of before adsorption), but HDLch was 17
mg/dl is 17 mg/dl (100%), albumin is 3.5
g/dl hardly decreased at 3.4 g/dl (97%),
Selectively adsorbed LDL.

Comparative example 1 Rohm &Haas's adsorbent
An adsorption experiment similar to that in Example 1 was conducted using a cation exchange resin IRC-50 having a COOH group (ion exchange capacity: 3.0 meq/ml, pore size: 200 to 2000 Å, particle size: 330 to 500 μm). As a result, LDL was 704mg/dl, but after adsorption it did not decrease much to 667mg/dl (95%), and HDLch was 17mg/dl to 15mg/dl.
(88%), albumin was 3.5g/dl and 3.1g/dl (89%).
%), and there was not much selectivity.

Comparative Example 2 D-glucosamitalonic acid (molecular weight 193, having one carboxyl group) was immobilized on CNBr-activated Sepharose 4B (manufactured by Pharmacia, Sweden) by a conventional method. The ion exchange capacity of the obtained adsorbent was 21 μeq/ml. When an adsorption experiment was conducted in the same manner as in Example 1, LDL was 704mg/dl.
67mg/dl (96%), HDLch 17mg/dl 16mg/dl
(94%), albumin 3.5g/dl
(100%), hardly any adsorption.

[Brief explanation of drawings]

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 material for adsorbing low-density lipoproteins, which has a polyanion moiety with a molecular weight of 600 or more on the surface, and has a negative charge density of 1 μeq/ml or more and 1 meq/ml or less. adsorbent. 2. The adsorbent for adsorbing low-density lipoproteins 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 lipoproteins 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.