JP3285441B2 - Adsorbent for lowering plasma viscosity of vasoocclusive disease plasma and apparatus for lowering plasma viscosity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an adsorbent and an apparatus for reducing plasma viscosity by adsorbing and removing high-molecular substances in plasma such as fibrinogen and low-density lipoprotein (hereinafter referred to as LDL). It is intended to be used for treatment of vasoocclusive disease in extracorporeal circulation treatment.

[0002]

2. Description of the Related Art Conventional vascular obstructive diseases have been conventionally treated by a method of forming blood vessels by a surgical operation or a percutaneous procedure using a catheter, an anticoagulant such as heparin, urokinase or plasminogen. -Medical methods using thrombolytic enzymes such as activators have been performed.
Contrast of the vascular occlusive disease, the arteriosclerosis obliterans and focal segmental glomerulosclerosis, covalently bound to dextran sulfate sulfated polysaccharide cellulosic porous beads LDL
Extracorporeal circulation treatment using an adsorbent has been attempted for the purpose of removing LDL. However, the adsorbent did not have the ability to adsorb fibrinogen and α2 macroglobulin, and was insufficient for the purpose of lowering plasma viscosity. Further, a double membrane filtration method using a membrane to separate by molecular weight has been attempted for the purpose of reducing the viscosity of plasma of an autoimmune disease patient by removing fibrinogen and low-density lipoprotein. However, the double membrane filtration method has problems such as the necessity of replacement fluid, lack of selectivity for decomposed substances, and extremely low removal efficiency.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an adsorbent and a plasma viscosity reducing device for efficiently reducing the viscosity of vasoocclusive disease plasma by extracorporeal circulation.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to a method for adsorbing fibrinogen and low-density lipoprotein by an adsorbent having a compound having a hydrophobic structure and a negative functional group on the surface thereof, thereby producing a blood vessel occlusive disease plasma. It is characterized by lowering the viscosity.

Object of the Invention The purpose of the apparatus for reducing plasma viscosity of the present invention is to reduce the plasma viscosity efficiently and ultimately to reduce the blood viscosity. Blood viscosity is determined by plasma viscosity and the flexibility and adhesiveness of red blood cells, white blood cells, or platelets when passing through a small space. Therefore, when the blood cell condition such as red blood cells and white blood cells greatly affects the blood viscosity, the present invention is not always suitably used. According to the study of the inventors, in autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue disease, the effect of blood cell status on blood viscosity is large, and thus is not always desirable for the purpose of the present invention. The plasma targeted by the plasma viscosity lowering device of the present invention is vaso-occlusive disease plasma, and specifically, macular degeneration such as maculopathia, obstructive arteriosclerosis, focal glomerulosclerosis, Acute progressive glomerulonephritis, obstructive thromboangiitis, thrombotic thrombocytopenic purpura,
Hemolytic uremic disease, hepatic venous thrombosis, hepatic artery thrombosis, venous obstructive disease sometimes seen in intrahepatic vein, portal vein obstructive disease, venous thrombosis, cerebrovascular disorder often seen in patients with central venous catheter insertion, etc. And plasma derived from the above diseases. Alternatively, it can be used for reducing the viscosity of plasma after percutaneous coronary angioplasty or cardiovascular bypass surgery, and is useful in preventing restenosis. Among them, macular degeneration, obstructive arteriosclerosis, focal glomerulosclerosis, acute progressive glomerulonephritis, obstructive thromboangitis, venous thrombosis, cerebrovascular disease, percutaneous coronary angioplasty or cardiovascular bypass It is more preferably used for postoperative plasma, and most preferably macular degeneration.

Substances to be adsorbed The substances to be adsorbed by the apparatus for reducing plasma viscosity of the present invention are fibrinogen and LDL. These plus very low-density lipoprotein (hereinafter referred to as V LDL) also it is preferred to adsorb.

Structure of Plasma Viscosity Reducing Apparatus The plasma viscosity reducing apparatus referred to in the present invention comprises an adsorbent made of a crosslinkable porous material having on its surface a compound having a hydrophobic structure and a negative functional group (hereinafter referred to as a ligand). , An inlet and an outlet, and means for preventing leakage of the adsorbent. This plasma viscosity reduction device is an inlet for blood,
Means for mixing an anticoagulant, means for perfusing blood, means for separating plasma from blood using a hollow fiber membrane, means for perfusing the separated plasma to a plasma viscosity reducing device, plasma viscosity reducing device, and plasma separation Means for mixing blood and plasma with reduced viscosity, and means for extracting blood can be used by being incorporated in a device having this order. Alternatively, a blood inlet, a means for mixing an anticoagulant, a means for perfusing blood, a plasma viscosity lowering device, and a means for deriving blood with reduced plasma viscosity may be incorporated in a device having this order and used. it can. Either type may be used, but it is particularly desirable to have a means for separating plasma from blood, since it has little effect on blood cell components such as platelets.

Ligand Properties The ligand used in the present plasma viscosity reducing device is preferably a compound having a negative functional group. The term "negative functional group" as used herein refers to a functional group that is negative at neutral pH, such as a carboxyl group, a sulfate group, and a phosphate group. Among them, a carboxyl group and a sulfate group are more preferably used in terms of LDL adsorption ability. Most preferred is a sulfate group. Although fibrinogen can be adsorbed even by a hydrophilic ligand having a large number of negative functional groups, in order to obtain a sufficient adsorption ability, the molecular weight of the ligand is 100,000 or more and at least one negative molecule per 200 molecular weight. It was preferred that functional groups were present. On the other hand, when the ligand has a high molecular weight, the stability may be reduced, the reactivity when the ligand is covalently bonded to the crosslinked porous material may be reduced, or blood kinin may be generated when it comes into contact with plasma in practical use. There were problems such as increasing. From the above points, it is more preferable to use a hydrophobic structure for fibrinogen adsorption. When the hydrophobic structure is expressed in the form of a substituent or a hydrocarbon, examples thereof include a chain alkyl structure such as methyl, ethyl, propyl, and butyl, and a cyclic structure such as benzene, cyclohexene, benzofuran, benzimidazole, and indole. Refers to a derivative. These may have other substituents. Among these, a cyclic structure and its derivative are particularly preferable from the viewpoint of adsorption ability.

The molecular weight of the ligand may be any. The molecular weight of the ligand is not particularly limited. However, the molecular weight is large for the adsorption of LDL and VLDL, and a large number of negative functional groups are contained in one ligand compound. It is preferable to have it because it can be easily bonded to LDL or the like at multiple points. On the other hand, when the molecular weight is large, the ligand is difficult to extend from the crosslinked porous body for a long time due to the hydrophobic structure, and the effect of the molecular weight is not exhibited. Therefore, a preferable molecular weight is 200 or more and 50,000 or less. The density of the negative functional group in the ligand is preferably one or more per 600 molecular weight, and more preferably one or more per 400 molecular weight in terms of LDL adsorption ability. In addition, one or more hydrophobic structures per 600 molecular weight, more preferably one or more per 400 molecular weight.

Specific Examples of Ligands Preferable specific examples of the above-mentioned ligands include styrenesulfonic acid, dodecylbenzenesulfonic acid, β-
Synthetic compounds such as methacryloyloxyethyl hydrogen phthalate, 2-acryloyloxyethyl phthalic acid, hesperitic acid, vinylbenzoic acid, 4-phosphonylstyrene, and adenosine-3'-phosphate, guanosine-2'-phosphate, Nucleic acids such as cytidine-2'-phosphate and thymidine-3'-phosphate, amino acids such as ergothioneine, kynurenine, phenylalanine, phenylglycine, tryptophan, tyrosine and thyroxine and derivatives thereof, or oligomers or polymers containing these as polymerized units And the like. In the case of an amino acid or its oligomer or polymer, the carboxyl group in the monomer may be used in the reaction during the polymerization,
Upon polymerization, it is desirable to copolymerize with an amino acid having two or more negative functional groups such as a monoaminodicarboxylic acid type amino acid, or a derivative thereof. Examples of these amino acids include glutamic acid, aspartic acid, cysteic acid and the like. Among the above, more preferred examples include monomers, oligomers and polymers of styrenesulfonic acid and vinylbenzoic acid, and monomers of tryptophan, and copolymers with other monomers containing these as monomers. More preferred are polystyrene sulfonic acid and polyvinyl benzoic acid, and most preferred is polystyrene sulfonic acid.

Crosslinked Porous Material The crosslinked porous material referred to in the present invention is a material which is usually used as a carrier of an adsorbent for immobilizing a ligand, and which is crosslinked from the viewpoint of plasma circulation. Quality material. As the shape of the crosslinked porous material, spherical, granular, fibrous, hollow fiber, flat membrane and the like are all effectively used,
Spherical or granular forms are more preferably used in terms of plasma distribution in practical use. A spherical or granular average particle diameter of 10 μm to 2500 μm is easy to use, but 25 μm to 10 μm.
It is preferably in the range of 00 μm, more preferably 50 μn to 600 μm. The crosslinked porous material preferably has an exclusion limit molecular weight of 5 × 10 ⁵ or more and 1 × 10 ⁸ or less. Further, from the viewpoint that high adsorption performance of VLDL can be exhibited, those having 2 × 10 ⁶ or more and 2 × 10 ⁷ or less are more preferable. If the exclusion limit molecular weight is less than 2 × 10 ⁶ , VL
If the adsorbing capacity of DL decreases, and if it is less than 5 × 10 ⁵ , the adsorbing capacity of LDL also decreases, and if it exceeds 1 × 10 ⁸ , the pore size becomes too large, and the surface area contributing to adsorption decreases. , VLDL and fibrinogen adsorption capacity is reduced.

Materials of the crosslinked porous material Specific examples of the crosslinked porous material include, for example, one or more of methyl methacrylate, polyvinyl alcohol, styrene, divinylbenzene, vinyl ether, maleic anhydride, polyamide, etc. A synthetic polymer,
And / or a hard gel obtained by crosslinking and synthesizing a natural polymer such as cellulose as a raw material. These are hydroxyethyl methacrylate, a polymer material having a hydroxy group such as hydroxyethyl acrylate, vinylamine,
Copolymers of monomers such as dimethylaminoethyl (meth) acrylate and other polymerizable monomers, block copolymers such as segmented polyurethane and segmented polyester, and monomers having polyethylene oxide chains and others And a coating layer such as a graft copolymer such as a copolymer with a polymerizable monomer. Among these, gels of crosslinked synthetic cellulose such as Cellulofine and synthetic polymers made of polyvinyl alcohol are hard and active groups can be obtained relatively easily on the gel surface. Above.

Method for Introducing Ligand into Cross-Linked Porous Material In order to introduce ligand into the cross-linked porous material, coating is performed by immersing the cross-linked porous material in a liquid in which the ligand is dissolved or spraying the liquid. Method, a method of covalently bonding to the surface of the crosslinked porous material by a grafting method chemically or by using radiation or an electron beam, or a method of covalently bonding to the surface of the crosslinked porous material through a functional group by a chemical method. is there. Among them, the grafting method and the covalent bonding method via a functional group are preferable because there is no danger of elution of the ligand during use. When the crosslinked porous material has a coating layer, it can be insolubilized on the surface of the coating layer. The active group on the surface of the crosslinked porous material may be any as long as it can be substituted and / or added with a nucleophilic reactive group having an active hydrogen such as an amino group, a hydroxyl group or a carboxyl group in the ligand. As an example of a method for obtaining an active group in a crosslinked porous material, a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a bromoacetyl bromide method, and the like are known. Specific examples of the active group of the crosslinked porous material include an amino group, a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride group, a succinylimide group, a chlorine group, an aldehyde group, an amide group, an epoxy group, and a tresyl group. Can be Among them, an epoxy group derived by the epichlorohydrin method is particularly preferable because of its stability during heat sterilization.

Amount of negative functional group on the surface of the adsorbent If the amount of the negative functional group on the surface of the adsorbent is too large, the Hageman factor and prekallikrein are activated upon contact with plasma to accelerate the decomposition of high molecular weight kininogen. May cause elevated blood kinin levels. On the other hand, if the amount of the negative functional group is too small, the increase in blood kinin concentration can be sufficiently suppressed, but the adsorption ability of the substance to be adsorbed cannot be sufficiently obtained,
After all it is not desirable. The amount of the preferred negative functional group on the surface of the adsorbed material per swollen adsorbent volume, at which the present invention exhibits the best effect, is 0.5% when measured by a neutral salt decomposition method.
It is not less than μ equivalent / ml and not more than 100 μ equivalent / ml. Particularly, when the amount is 1.0 μequiv / ml or more and 50 μequiv / ml or less, the most preferable result is obtained.

[0015]

Example 1 Porous beads made of polyvinyl alcohol were used as a crosslinked porous material. The porous beads had an exclusion limit molecular weight of 5 × 10 ⁶ and a swollen particle size of 70 μm.
One volume of the porous beads and 5 volumes of a 10% aqueous ethanol solution in which 5% of sodium styrenesulfonate is dissolved are placed in a glass container, irradiated with 25 KGy of γ-ray, and radiation-grafted, and polystyrenesulfonic acid is used as a ligand on the surface as a ligand. An adsorbent was obtained. The amount of negative functional groups on the surface of the obtained adsorbent was determined by the neutral salt decomposition method.
It was 25.4 μ equivalent per liter. The molecular weight of the immobilized ligand was 44,000 when the molecular weight of polystyrenesulfonic acid in the aqueous ethanol solution after grafting was measured by high performance liquid chromatography using polyethylene glycol as a control. 5 ml of the adsorbent was filled into a cylindrical container having an inlet and an outlet and having a built-in mesh for preventing the adsorbent from leaking, to obtain a plasma viscosity reducing device. The macular degeneration plasma obtained using heparin as an anticoagulant is perfused into the plasma viscosity lowering apparatus at a plasma perfusion rate of 0.3 ml / min using a peristaltic pump, and 5 ml of plasma flowing out of the plasma viscosity lowering apparatus is discharged. Collected as one fraction. The first fraction was discarded, and the second to fourth fractions were collected, and the concentrations of LDL, VLDL, and blood kinin in each fraction were measured.
At this time, the second to fourth fractions were mixed, and the plasma viscosity was measured. In a similar manner, macular degeneration plasma obtained using citric acid as an anticoagulant was perfused, and the fibrinogen concentration in each fraction was measured. The concentrations of LDL and VLDL were determined by specific gravity centrifugation, and the concentrations of fibrinogen were determined by colorimetry.
The ratio of the decrease in the average value of the concentration from the fraction to the fourth fraction with respect to the original plasma concentration was determined as the adsorption rate. The concentration of each fraction of blood kinin was measured by radioimmunoassay, and the highest concentration from the second fraction to the fourth fraction was defined as the blood kinin concentration. The plasma viscosity was determined as a relative viscosity to water using an Oswald viscometer. LDL, VLDL in original plasma,
The concentrations of fibrinogen and blood kinin were 55
6 mg / dl, 109 mg / dl, 167 mg / dl,
It was 42.2 pg / ml. The relative viscosity is 2.06
Met. LDL, VLD after perfusion with this plasma viscosity lowering device
The L adsorption rates were 97.5% and 90.8%, respectively, and fibrinogen was below the detection limit and the adsorption rate was 88.0% or more. This plasma viscosity lowering device had high adsorption capacity for LDL, VLDL, and fibrinogen. On the other hand, blood kinin was 73.1 pg / ml and was not increased. At this time, the plasma viscosity was 1.56, and the rate of decrease in plasma viscosity was 24.3%, indicating an excellent function of decreasing plasma viscosity.

[0016]

Example 2 In the same manner as in Example 1 except that sodium styrene sulfonate was changed to vinylbenzoic acid and the concentration of vinylbenzoic acid used during grafting was 7%, polyvinylbenzoic acid was used as a ligand on the surface. An adsorbent was obtained. The amount of the negative functional group on the surface of the obtained adsorbent was determined by the neutral salt decomposition method, and was 34.2 μ equivalent per 1 ml of the adsorbent. The molecular weight of the immobilized ligand was 29,000 as measured by measuring the molecular weight of polyvinyl benzoic acid in the aqueous ethanol solution after grafting. Acutely advanced glomerulonephritis plasma was perfused in the same manner as in Example 1 using this adsorbent. LD in original plasma
The concentrations of L, VLDL, fibrinogen, and blood kinin were 532 mg / dl, 101 mg / dl, and 17 respectively.
It was 3 mg / dl and 31.5 pg / ml. The relative viscosity was 1.88. The adsorption rates of LDL and VLDL after perfusion with the plasma viscosity lowering device were 97.4% and 9%, respectively.
0.1%, fibrinogen was below the detection limit, and the adsorption rate was 88.4% or more. This plasma viscosity reduction device is LD
It had a high adsorption capacity for L, VLDL and fibrinogen. On the other hand, blood kinin was 67.5 pg / ml and was not increased. The plasma viscosity at this time was 1.3
8. The rate of decrease in plasma viscosity was 26.5%, indicating an excellent function of decreasing plasma viscosity.

[0017]

Example 3 An epoxy group was introduced into the porous beads used in Example 1 by using epichlorohydrin in dimethyl sulfoxide. The epoxy equivalent of the epoxy group-introduced porous beads obtained at this time was 116.0 μ / ml of adsorbent.
eq. The epoxy group-introduced porous beads and tryptophan were reacted under acidic conditions to covalently bond the epoxy group and the amino group of tryptophan, thereby obtaining an adsorbent using free tryptophan having a carboxyl group as a ligand. The amount of the negative functional group on the surface of the obtained adsorbent was determined by the neutral salt decomposition method, and was 40.8 μ equivalent per 1 ml of the adsorbent. Example 1 using this adsorbent
The macular degeneration plasma was perfused in the same manner as described above. The adsorption rate of LDL and VLDL after perfusion with this plasma viscosity lowering device was 9 respectively.
0.6%, 84.4%, and fibrinogen were below the detection limit, and the adsorption rate was 88.0% or more. This plasma viscosity lowering device had high adsorption capacity for LDL, VLDL, and fibrinogen. On the other hand, blood kinin is 55.7p
g / ml and was not elevated. At this time, the plasma viscosity was 1.60, and the reduction rate of the plasma viscosity was 22.3%, indicating an excellent function of lowering the plasma viscosity.

[0018]

Comparative Example 1 Macular degeneration was carried out in the same manner as in Example 1 using a commercially available LDL adsorbent (LA-15, manufactured by Kaneka Chemical Co., Ltd.) in which dextran sulfate having a molecular weight of 5,000 was insolubilized on a crosslinked cellulose carrier. Plasma was perfused. The adsorption rates of LDL, VLDL, and fibrinogen after perfusion with the present plasma viscosity lowering apparatus were 90.1%, 83.5%, and 6.6%, respectively.
This plasma viscosity lowering device had the ability to adsorb LDL and VLDL, but did not have the ability to adsorb fibrinogen. On the other hand, blood kinin was activated at a very high level of 18,300 pg / ml, which was not preferable for practical use. At this time, the plasma viscosity was 2.04, the rate of decrease in plasma viscosity was 1.0%, and there was no function of decreasing plasma viscosity.

[0019]

Comparative Example 2 Instead of styrene sulfonic acid in Example 1, a mixture of styrene monomer and styrene sulfonic acid at a molar ratio of 6: 1 was used. The other steps were carried out in the same manner to obtain an adsorbent using a copolymer of styrene and styrenesulfonic acid as a ligand. From the aqueous ethanol solution after the reaction, the ligand molecular weight was 28,000. The fraction was collected and evaporated to dryness, and the sulfonic acid group was measured by titration. As a result, the density of the negative functional group in the ligand was one per 800 molecular weight. Macular degeneration plasma was perfused in the same manner as in Example 1 using this adsorbent. L after perfusion with the present plasma viscosity lowering device
The adsorption rates of DL, VLDL, and fibrinogen were 13.7%, 9.2%, and 88.0%, respectively. Although this plasma viscosity lowering device had the ability to adsorb fibrinogen, it did not have the ability to adsorb LDL and VLDL.
On the other hand, blood kinin hardly increased to 86.7 pg / ml because of the hydrophobic structure in the ligand. At this time, the plasma viscosity was 1.93, and the decrease rate of the plasma viscosity was 6.3.
%, And the function of lowering the plasma viscosity was very low.

[0020]

According to the plasma viscosity reducing device of the present invention, the viscosity of vasoocclusive disease plasma can be efficiently reduced.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-269203 (JP, A) JP-A-61-206457 (JP, A) JP-A-5-200111 (JP, A) JP-A-4-201 314456 (JP, A) JP-A-5-285381 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61M 1/36 540

Claims (6)

(57) [Claims] 1. A compound having a hydrophobic structure and a negative functional group
And the amount of the negative functional group is 0.5 μeq / m
An adsorbent for decreasing plasma viscosity of vascular occlusive disease plasma, wherein the viscosity of the plasma is reduced by removing fibrinogen and low-density lipoprotein from 1 to 100 μeq / ml . 2. The method according to claim 1 , wherein the compound has at least one compound per 600 molecular weight.
The above negative functional group and one or more sparse
The vascular closure according to claim 1, wherein the vessel has an aqueous structure.
Adsorbent for decreasing plasma viscosity of embolic disease plasma . 3. Vascular occlusive disease plasma is macular degeneration, obstructive arteriosclerosis, focal glomerulosclerosis, acute progressive glomerulonephritis, obstructive thromboangitis, venous thrombosis, cerebrovascular disease, The plasma viscosity of the vaso-occlusive disease plasma according to claim 1, which is plasma after any of percutaneous coronary angioplasty or cardiovascular bypass surgery.
Adsorbent for temperature reduction . 4. A compound having a hydrophobic structure and a negative functional group.
And the amount of the negative functional group is 0.5 μeq / m
An apparatus for lowering plasma viscosity of vasoocclusive disease plasma, which has a built-in adsorbent for adsorbing fibrinogen and low-density lipoprotein , which has a volume of 1 to 100 μeq / ml and has an inlet and an outlet for plasma. 5. One or more negative agents per 600 molecular weight
Functional groups and one or more hydrophobic structures per 400 molecular weight
The plasma viscosity lowering device according to claim 4, wherein a crosslinked porous material having a compound having the compound on the surface is packed as an adsorbent for fibrinogen and low-density lipoprotein. 6. Vascular occlusive disease plasma is macular degeneration, obstructive arteriosclerosis, focal glomerulosclerosis, acute progressive glomerulonephritis, obstructive thromboangitis, venous thrombosis, cerebrovascular disease, The plasma viscosity lowering device according to claim 4, which is any plasma after percutaneous coronary angioplasty or cardiovascular bypass surgery.