JP4783970B2 - Adsorbent for adsorption of oxidized low density lipoprotein - Google Patents

Description

【００４０】
【発明の効果】
本発明の吸着材は酸化ＬＤＬを選択的に吸着するため、体液中の他の成分をほとんど損なうことなく、酸化ＬＤＬを取り除くことができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an adsorbent for oxidized low density lipoprotein (oxidized LDL). More specifically, the present invention relates to an adsorbent for selectively removing oxidized LDL from body fluids and suppressing reduction or progression of various symptoms of arteriosclerosis.
[0002]
[Prior art]
Arteriosclerosis is a general term for lesions that show thickening of the arterial wall and have lost elasticity and have become hardened. Among them, the frequency is extremely high and the important disease is atherosclerosis, Generally speaking, arteriosclerosis often means atherosclerosis. It is lipid peroxide that plays an important role in the formation of atherosclerotic lesions. Among them, oxidized LDL has various biological actions, and in addition to the action of suppressing nitric oxide (NO) production from endothelial cells, monocytes are transported and accumulated in the subendothelium, and they themselves become macrophages. It plays an important role in the onset and progression of arteriosclerosis by taking oxidized LDL itself into foam cells and promoting plaque formation on the arterial wall, as well as promoting endothelial cell and smooth muscle cell damage. Therefore, it is desirable to remove lipid peroxide, particularly oxidized LDL, from the blood.
[0003]
Up to now, the only methods for treating arteriosclerosis have been to spread surgically constricted blood vessels or to inhibit LDL metabolism using drugs. The former is highly invasive and cannot prevent the progression of arteriosclerosis. The latter has a problem that it is not effective for arteriosclerosis patients without LDL metabolic abnormality.
[0004]
[Problems to be solved by the invention]
The present invention provides an adsorbent capable of selectively adsorbing oxidized LDL with almost no loss of active ingredients in body fluids in order to solve the above problems.
[0005]
[Means for Solving the Problems]
The object of the present invention is achieved by a lipid peroxide adsorbent characterized in that an alkylated polyalkyleneimine having an alkyl group having 2 to 16 carbon atoms is imparted to a support.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
[0007]
The adsorbent of the present invention is characterized in that an alkylated polyalkyleneimine having a selective affinity for oxidized LDL is imparted to a support.
[0008]
In the case of hyperlipidemia patients, the amount of LDL in the blood is large, so removing LDL is effective in preventing the progression of arteriosclerosis, but in the case of dialysis patients and patients with heart disease, The LDL concentration in the medium is often at the same level as healthy individuals. Moreover, since high-density lipoprotein (HDL) has a function as an arteriosclerosis protective factor, the HDL concentration in the blood of dialysis patients and patients with heart disease should not be lowered. Thus, it is desirable to selectively remove lipid peroxide, particularly oxidized LDL, from dialysis patients and patients with heart disease. In the present invention, when the adsorbent and plasma interact with each other under the condition that the plasma volume per 1 m ^{2 of} the support surface area is 3.3 × 10 ² ml / m ² , the initial amount contained in the plasma The adsorption removal rate of oxidized low density lipoprotein with a concentration of 2 μg / ml is 60% or more, the adsorption removal rate of low density lipoprotein is less than 20%, and the adsorption removal rate of high density lipoprotein is less than 15%. It can be used as an index of selectivity.
[0009]
The support in the present invention refers to a substance that does not dissolve in water for providing an alkylated polyalkylenimine. The support used in the present invention may be either organic or inorganic. Specific examples of the material of the support include aromatic polyvinyl compounds represented by polystyrene, polysulfone, polyetherimide, polyamide, polyimide, polyether, polyphenylene sulfide, and the like, but other body fluid components other than the target oxidized LDL Polysulfone which is difficult to adsorb is preferred. The term “body fluid” as used herein refers to blood, ascites, lymph fluid, intra-articular fluid, other biological components derived from living bodies, and fractional components obtained therefrom.
[0010]
As a method for imparting the alkylated polyalkyleneimine to the support, a physical adsorption method, an ionic bond method, a covalent bond immobilization method, or the like can be used. However, since it is important that the alkylated polyalkyleneimine is not desorbed and eluted from the support for the storage stability and stability of the adsorbent, it is desirable that the adsorbent be applied by a covalent bond method capable of strong fixation. . That is, it is advantageous that an amine-bonding group that can be used for the immobilization reaction of the alkylated polyalkyleneimine is present on the surface of the support. Specific examples of such an amine bond group include a halomethyl group, a haloacetyl group, a haloacetamidomethyl group, a halogenated alkyl group, an epoxide group, a carboxyl group, an isocyanate group, a thioisocyanate group, and an acid anhydride group. can give. In particular, the active halogen group is preferably used in the present invention because it is easy to produce and has high reactivity, and in addition to being able to perform the immobilization reaction under mild conditions, the resulting covalent bond is chemically stable.
[0011]
Furthermore, specific examples of the support material having an amine bond group include chloroacetamidomethyl polystyrene, chloroacetamidomethylated polysulfone, and chloroacetamidomethylated polyetherimide. Furthermore, these are preferable because they are soluble in an organic solvent because they are easy to mold. Of these, chloroacetamidomethylated polysulfone is particularly preferred.
[0012]
When the material of the support is a polymer compound, the molecular weight is preferably 5000 or more and 1,000,000 or less, particularly 10,000 or more and 200,000 or less.
[0013]
The alkylated polyalkyleneimine as referred to in the present invention is obtained by alkylating a part of the nitrogen atom of the polyalkyleneimine. Alkylation can be performed by alkylating polyalkyleneimine with a halogenated hydrocarbon typified by butyl bromide, octyl bromide, lauryl bromide or the like alone or as a mixture. The alkyl group may be linear or branched. Polyalkyleneimine is preferably polyethyleneimine because of its availability. Such an alkylated polyalkyleneimine varies in adsorption performance depending on the number of carbon atoms in the alkyl group and the alkylation rate.
[0014]
In the alkylated polyalkyleneimine of the present invention, the alkyl group has 2 to 16 carbon atoms. When the number of carbon atoms of the alkyl group decreases, the oxidized LDL adsorption ability decreases. Alkyl halides having 20 or more carbon atoms are generally expensive and have low selectivity for oxidized LDL. The number of carbon atoms of the alkyl group is particularly preferably 4 or more and 12 or less.
[0015]
In the alkylated polyalkyleneimine, if the alkylation rate is too small, the adsorptive capacity is lowered, and if it is too large, the selectivity to oxidized LDL is lowered. Therefore, the alkylation rate is preferably 20% or more and 50% or less.
[0016]
In the alkylated polyalkyleneimine, if the average degree of polymerization of the polyalkyleneimine is too small, the adsorptive capacity decreases, and if it is too large, the selectivity to oxidized LDL is decreased, and therefore, 40 to 2500 is preferable, and 200 to 700 is particularly preferable.
[0017]
The alkylated polyalkyleneimine used in the present invention can be easily prepared by mixing a halogenated hydrocarbon such as an alkyl bromide with a polyalkyleneimine solution at a temperature of room temperature to 100 ° C. it can. This reaction proceeds quantitatively with respect to the charged amount even at room temperature if the alkylation rate is 50% or less in a polar solvent such as ethyl alcohol, isopropyl alcohol, tetrahydrofuran and dimethylformamide.
[0018]
To produce an adsorbent for oxidized LDL, a homogeneous reaction method in which a support and an alkylated polyalkyleneimine are reacted in a solution, and a heterogeneous system in which an alkylated polyalkyleneimine solution is brought into contact with the molded article of the support There is a method of reaction.
[0019]
As an example of the method by homogeneous reaction, it can be easily synthesized by adding an appropriate amount of an alkylated polyalkylenimine to a solution of chloroacetamidomethylated polysulfone and reacting at a temperature of 0 to 100 ° C. At this time, if the alkylation rate of the alkylated polyalkyleneimine is high, the alkylated polyalkyleneimine hardly dissolves in the solvent in which the chloracetamidomethylated polysulfone is dissolved. Further, when an unalkylated polyalkyleneimine is reacted with chloroacetamidomethylated polysulfone, gelation tends to occur. Therefore, it is convenient to obtain a target alkylation rate by introducing an alkylated polyalkyleneimine having a low alkylation rate and then adding further alkyl bromide. In addition, as a solvent for the reaction in a homogeneous system, tetrahydrofuran, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, methylpyrrolidone and the like are preferably used.
[0020]
In the present invention, a method for surface treatment of the support in a heterogeneous system is also possible. For this purpose, a solvent that does not dissolve the support but dissolves the alkylated polyalkyleneimine is preferably used.
[0021]
As an example of the heterogeneous reaction, a molded product such as a fiber or hollow fiber of chloroacetamidomethylated polysulfone is immersed in an isopropanol solution of an alkylated polyalkyleneimine, and is easily produced by reacting at a temperature of 0 to 100 ° C. can do.
[0022]
The adsorbent of the present invention can be packed in an extracorporeal circulation column or the like and used for extracorporeal circulation treatment. That is, it can be directly molded into a fiber shape or a film shape by a uniform reaction, or fixed to a molded product by coating, or can be fixed to a fiber, a hollow fiber, a bead or the like by a non-uniform reaction. By immobilizing alkylated polyalkyleneimines in hollow fibers for artificial kidneys, in addition to the conventional function of removing uremic toxins and water, a new function of removing oxidized LDL in body fluids can be added. preferable. In particular, it has been confirmed that patients with long-term hemodialysis have low blood antioxidant activity and high levels of lipid peroxide, which may be attributed to arteriosclerosis in long-term dialysis patients. Since sexual diseases and the like are increasing, it is preferable to use the adsorbent of the present invention for the purpose of removing oxidized LDL in the body fluid of dialysis patients.
[0023]
Hereinafter, the performance measurement conditions of the membrane type adsorber of the present invention will be described.
(1) Production of Antioxidant LDL Antibody The one produced by Plate et al. Was used (H. Itabet et al., J. Biol. Chem. 269: 15274, 1994). That is, human atherosclerotic lesion homogenate was injected into mice for immunization, hybridomas were prepared from the spleens of the mice, and those that reacted with copper sulfate-treated LDL were selected. The antibody class is mouse IgM and does not react with untreated LDL, acetyl LDL, or malondialdehyde LDL. Reacts with several phosphatidylcholine peroxidation products, including aldehyde derivatives of phosphatidylcholine and hydroperoxides. What was dissolved in 10 mM borate buffer (pH 8.5) containing 150 mM NaCl was used (protein concentration 0.60 mg / ml).
(2) Preparation of oxidized LDL Commercially available LDL (manufactured by Funakoshi) was desalted and diluted with a phosphate buffer (hereinafter referred to as PBS) to a concentration of 0.2 mg / ml. % Was added and reacted at 37 ° C. for 16 hours. An oxidized LDL preparation was prepared by adding 25 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) to a concentration of 1 wt%, 10 wt% sodium azide to 0.02 wt%.
(3) Operation of adsorption experiment The above-mentioned oxidized LDL was added to healthy human plasma (Japanese, 30 years old, LDL (β-lipoprotein) concentration 275 mg / dl, HDL-cholesterol concentration 70 mg / dl) to 2 μg / ml.
[0024]
A support having a surface area of 9 cm ² was allowed to stand in 0.3 ml of the plasma at 37 ° C. for 4 hours. (The plasma volume per 1 m ^{2 of the} support surface area is 3.3 × 10 ² ml / m ² ).
[0025]
By quantifying the concentrations of oxidized LDL, LDL, and HDL in the plasma before and after interaction with the membrane, the respective adsorption removal rates were calculated by the following formula.
[0026]
Each adsorption removal rate (%) = 100 × (concentration before interaction−concentration after interaction) / concentration before interaction (4) Measurement of oxidized LDL, LDL, HDL concentration Antioxidant LDL antibody in PBS at 5 μg The solution was diluted to 100 ml / well in a 96-well plate, shaken at room temperature for 2 hours, and adsorbed on the wall at 4 ° C overnight or longer.
[0027]
Discard the antibody solution in the well and dispense 200 μl / well of Tris-HCl buffer (pH 8.0) containing 1 wt% Bovine Serum Albumin (BSA, “Fraction V”, Seikagaku Corporation) for 2 hours at room temperature. After blocking the wall by shaking, the BSA solution in the well is discarded, and plasma containing oxidized LDL and a standard for preparing a calibration curve (PBS buffer containing 0-2 μg / ml oxidized LDL) are added at 100 μl / well. Dispensed. Then, after shaking at room temperature for 30 minutes, it was left at 4 ° C. overnight.
[0028]
After returning to room temperature, the solution in the well was discarded, and the well was washed three times with Tris-HCl buffer (pH 8.0) containing 0.05 wt% “Tween-20” (Katayama Chemical). 100 μl / well of sheep anti-apo B antibody (THE BINDING SITE) diluted 2000 times with PBS was dispensed into the washed wells and shaken at room temperature for 2 hours, and then the anti-apo B antibody in the wells was discarded. The wells were washed three times with Tris-HCl buffer (pH 8.0) containing 05 wt% Tween-20. 100 μl / well of alkaline phosphatase-labeled donkey anti-sheep IgG antibody (CHEMICON) diluted 2000-fold with Tris-HCl buffer (pH 8.0) containing 2 wt% “Block Ace” (Dainippon Pharmaceutical) in the washed wells Poured and shaken for 2 hours at room temperature. Thereafter, the labeled antibody in the well is discarded, and the well is washed three times with Tris-HCl buffer (pH 8.0) containing 0.05 wt% “Tween-20”, and further, Tris-HCl buffer (pH 8.0). And washed twice. Subsequently, a 1 mg / ml solution (0.0005M MgCl ₂ , 1M diethanolamine buffer, pH 9.8) of p-nitrophenyl phosphate (Boehringer Mannheim GmbH) was dispensed at 100 μl / well and reacted at room temperature for an appropriate time. Then, the absorbance at 415 nm was measured with a plate reader. A calibration curve was drawn from the standard results to determine the oxidized LDL concentration.
[0029]
LDL was quantified using a β-lipo assay kit (Wako Pure Chemical Industries).
[0030]
Quantification of HDL was performed using an HDL-C measurement kit (Wako Pure Chemical Industries).
[0031]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to such an Example.
[0032]
Example 1
(1) Preparation of support After cooling a mixed solution of nitrobenzene and sulfuric acid to 0 ° C., methylol-α-chloroacetamide was added and dissolved, and this was stirred well into a nitrobenzene solution of “Udel” (polysulfone) at 10 ° C. Added while. This was stirred at room temperature for 3 hours. The reaction mixture was then placed in a large excess of cold methanol to precipitate the polymer. The precipitate was washed well with methanol and dried to obtain α-chloroacetamidomethylated polysulfone.
[0033]
Next, this α-chloroacetamidomethylated polysulfone was dissolved in DMAc so as to be 20 wt% at room temperature. Then, the solution was applied to a glass plate with a 0.1 mm spacer, and the flat film stretched with a doctor blade was immersed in water (room temperature) and solidified.
(2) α-Chloracetamidomethylated polysulfone flat membrane washed thoroughly with water applied to the support of N-butylated polyethyleneimine, a polyethyleneimine / isopropanol solution (polyethylene) having an average molecular weight of 10,000 (average degree of polymerization 233) Polyethyleneimine was imparted to the flat membrane by a covalent bond by immersing in an imine concentration of 5 wt% and stirring at room temperature for 24 hours. Thereafter, N-butyl bromide was added so that the butylated rate was 30%, and the mixture was stirred at room temperature for 24 hours to introduce N-butyl groups into polyethyleneimine. After completion of the reaction, the flat membrane was thoroughly washed with isopropanol and then subjected to an adsorption experiment.
[0034]
Example 2
In the same manner as in Example 1, a flat film to which laurylated polyethyleneimine having an average molecular weight of 10,000 (average polymerization degree of 233) and a laurylation ratio of 30% was formed as polyethyleneimine was subjected to an adsorption experiment.
[0035]
The experimental results are shown in Table 1.
[0036]
Comparative Example 1
An adsorption experiment was conducted in the same manner as in Example 1 on an α-chloroacetamidomethylated polysulfone flat membrane not provided with an alkylated polyethyleneimine.
[0037]
Comparative Example 2
An adsorption experiment was conducted in the same manner as in Example 1 on a flat film provided with polyethyleneimine having an average molecular weight of 10,000 (average polymerization degree 233) that was not alkylated.
[0038]
Table 1 shows the results of adsorption removal rates of oxidized LDL, LDL, and HDL in Examples 1 and 2 and Comparative Examples 1 and 2.
[0039]
[Table 1]

[0040]
【The invention's effect】
Since the adsorbent of the present invention selectively adsorbs oxidized LDL, the oxidized LDL can be removed with almost no loss of other components in the body fluid.

Claims (6)

An alkylated polyethyleneimine having an alkyl group having 4 to 12 carbon atoms and an alkylation rate of 20% to 50% is added to the support containing the polysulfone having an amine bond group in the molded article of the support. polyalkyleneimines solution you characterized in that it is provided by heterogeneous reaction of contacting the oxidized low-density lipoprotein adsorbing adsorbent. The adsorbent for oxidized low-density lipoprotein adsorption according to claim 1, wherein the average degree of polymerization of the polyalkyleneimine in the alkylated polyalkyleneimine is 40 or more and 2500 or less. When the amount of plasma per 1 m ^{2 of the} material is 3.3 × 10 ² ml / m ² , when the material interacts with the plasma, the initial concentration contained in the plasma is 2 μg / ml. The adsorbent for adsorbing oxidized low-density lipoprotein according to claim 1 or 2, wherein the adsorption removal rate of density lipoprotein is 60% or more. The adsorbent for adsorbing oxidized low-density lipoprotein according to any one of claims 1 to 3, wherein the adsorption removal rate of the low-density lipoprotein is less than 20% under the conditions according to claim 3. The adsorbent for adsorbing oxidized low-density lipoprotein according to any one of claims 1 to 3, wherein the adsorption removal rate of high-density lipoprotein is less than 15% under the conditions of claim 3. The adsorbent for oxidizing low-density lipoprotein adsorption according to any one of claims 1 to 5, wherein the amine-binding group is an α-chloroacetamidomethyl group.