JP2008086558A - Ldl absorbent material and ldl removing material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an LDL absorbent material capable of easily fixing an LDL absorption ligand under a mild condition even to a carrier such as a porous hollow fiber or a porous bead composed of polypropylene, polystyrene or polysulfone or the like without an active functional group. <P>SOLUTION: The LDL absorbent material is composed of an anionic polymer material. The polymer is water soluble at a temperature lower than a prescribed temperature (T) lower than a living body temperature and is poorly water soluble at a temperature higher than the prescribed temperature (T). For the polymer material, to a homopolymer composed of a star type polymer for which a compound having four or more N,N-dialkyl-dithiocarbamyl methyl molecular groups is an iniferter and light irradiation living polymerization of the methacrylic acid to it is performed, N-isopropyl acrylamido is block polymerized, and H of the methacrylic acid is replaced with Na thereafter. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an LDL adsorbent for removing LDL from blood and an LDL remover coated with the LDL adsorbent. In the present specification, LDL includes VLDL.

  It is known that lipoproteins present in blood, especially LDL, contain a large amount of cholesterol and cause arteriosclerosis. As a method for removing LDL from body fluids such as blood, a removal method using an adsorbent has been conventionally employed. As the adsorbent, a substance in which a ligand such as heparin or dextran sulfate exhibiting affinity for LDL is immobilized on a porous carrier has been put into practical use. Examples of the porous carrier include those made of polysaccharides such as crosslinked agarose and crosslinked chitosan, and silica gel.

  As a method for immobilizing a ligand on a carrier, there is a method using a functional group on the surface of the carrier, and the material itself has a functional group, or a treatment for introducing an active functional group into the carrier is performed. Examples of functional groups include amino groups, carboxyl groups, hydroxyl groups, and epoxy groups. Ligand fixation is generally covalently introduced so that the ligand is not detached during sterilization or blood treatment. .

For example, Japanese Patent Publication No. 6-11330 discloses epoxidation by treating crosslinked agarose with saturated NaOH and epichlorohydrin, and treating this with concentrated aqueous ammonia to introduce an amino group, which contains an aqueous solution containing sodium dextran sulfate. It is described that a ligand is introduced by adding 2 ml and adjusting the pH to 12.
JP 6-11330

  In the above Japanese Patent Publication No. 6-11330, the carrier must be treated under harsh conditions when introducing the ligand, which limits the porous material that can be used as the carrier. There is also a risk that the active substance used in the introduction reaction may remain or be eluted. Furthermore, it is not possible to inactivate the active sites (for example, amino groups introduced into polysaccharides or residual silanol groups OH groups of silica gel, etc.) after introduction of ligands into all reaction active sites and after-treatment. There is also a problem that these polar functional groups activate complement C3a in blood.

  The present invention is an LDL adsorption capable of easily immobilizing an LDL adsorption ligand under mild conditions even on a carrier such as porous hollow fiber and porous beads made of polypropylene, polystyrene, polysulfone, etc. having no active functional group. The purpose is to provide materials.

  The LDL adsorbent of the present invention is an LDL adsorbent made of a polymer material, and the polymer material is water-soluble at a temperature lower than a predetermined temperature (T) lower than the living body temperature, and from the predetermined temperature (T). Furthermore, it is characterized by poor water solubility at high temperatures.

  This polymer material is a block copolymer of a homopolymer and an N-alkylacrylamide such as N-isopropylacrylamide, and the homopolymer block is preferably anionic.

  The polymer material is preferably anionic by bringing the block copolymer into contact with an aqueous alkali solution such as an aqueous NaOH solution.

  The molecular weight of this homopolymer is preferably 500 to 300,000.

  The molecular weight of this block copolymer is preferably 3,000 to 500,000.

  The predetermined temperature (T) is preferably a temperature between 24 and 26 ° C.

  This homopolymer is a star polymer in which a compound having 4 or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and a vinyl monomer is photoirradiated by living polymerization. Is preferred.

  The compound having four or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and four or more N, N-dialkyl-dithios as branched chains in this nucleus. Those having a carbamylmethyl molecular group bonded thereto are preferred.

  The vinyl monomer is preferably acrylic acid or methacrylic acid, particularly methacrylic acid.

  The LDL removing material of the present invention is such that the LDL adsorbent of the present invention is supported on a carrier.

  The LDL adsorbent of the present invention is water-soluble at a temperature lower than the predetermined temperature. This is because the polymer chain of N-alkylacrylamide such as N-isopropylacrylamide constituting a part of the LDL adsorbent of the present invention is hydrophilic at low temperature and becomes hydrophobic at high temperature. Due to having. Therefore, the LDL adsorbing material of the present invention is dissolved in water at a temperature lower than the predetermined temperature and adhered to the carrier for the LDL removing material, and then the temperature is higher than the predetermined temperature. And is supported on the carrier.

  The LDL adsorbent can be easily fixed to a carrier such as a porous hollow fiber or porous bead made of polypropylene, polystyrene, polysulfone or the like having no active functional group, under mild conditions.

  This LDL adsorbent is poorly water-soluble at a living body temperature and maintains a state of being a carrier. Therefore, the LDL adsorbent does not flow out of the carrier even when blood at a biological temperature is brought into contact with the LDL remover of the present invention.

  The LDL adsorbent of the present invention is made of a polymer material that is water-soluble at a temperature lower than a predetermined temperature (T) lower than the living body temperature and hardly water-soluble at a temperature higher than the predetermined temperature (T).

  The polymer material is a block copolymer of a homopolymer and an N-alkylacrylamide such as N-isopropylacrylamide, and the homopolymer block is preferably anionic.

  As this homopolymer, a star polymer in which a compound having 4 or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and a vinyl monomer is light-irradiated by living polymerization to this is used. It is.

  In this specification, the iniferter is a polymerization initiator that generates radicals upon irradiation with light, a function as a chain transfer agent, a function that bonds with the growth terminal to stop the growth, and a polymerization when light irradiation stops. It is a molecule that functions as a polymerization initiator / termination agent for stopping.

  As a compound having 4 or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule, 4 or more N, N-dialkyl-dithiocarbamylmethyl molecular groups are bonded as a branched chain to the benzene ring. These are preferred, and specific examples are as follows. That is, the 4-branched chain is obtained by addition reaction of 1,2,4,5-tetrakis (bromomethyl) benzene and sodium N, N-dithiocarbamate (sodium N, N-dithiocarbamate) in ethanol. 1,2,4,5-tetrakis (N, N-dithiocarbamylmethyl) benzene, and as a 6-branched chain, addition reaction of hexakis (bromomethyl) benzene and sodium N, N-dithiocarbamate in ethanol Hexakis (N, N-dithiocarbamylmethyl) benzene obtained by the above-mentioned process.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. The nonpolar solvent is preferably an alkyl halide or an alkylene halide, and particularly preferably chloroform or methylene chloride, especially chloroform.

  As a vinyl monomer for polymerizing the iniferter to form a hydrophobic homopolymer, acrylic acid or methacrylic acid, particularly methacrylic acid is preferable.

  To react the iniferter with the vinyl monomer, prepare a raw material solution containing the iniferter and the vinyl monomer, and irradiate it with light to produce a reaction product in which the vinyl monomer is bound to the iniferter. Let

  The concentration of the vinyl monomer in the raw material solution is preferably 70% by weight or more, for example, 70 to 99% by weight.

  The concentration of the iniferter is preferably about 10 to 1000 mM / L.

The wavelength of the irradiated light is preferably 240 to 300 nm. The irradiation time of the light depends on the irradiation intensity, about 1 to 60 minutes are preferred, about 1 to 30 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  By this light irradiation, the target branched star polymer (hydrophobic homopolymer) is produced in the reaction solution, so that it is purified as necessary to obtain a homopolymer composed of the branched star polymer.

  The molecular weight of the branched star polymer depends on the number of branched chains, but is preferably 3,000 to 500,000, particularly 5,000 to 200,000, particularly 10,000 to 100,000.

  N-alkylacrylamide (the alkyl group preferably has 1 to 10 carbon atoms), particularly preferably N-isopropylacrylamide is block copolymerized with the homopolymer composed of the branched star polymer thus produced. A polymer material is used. The polymer chain of the N-alkylacrylamide has a temperature dependency that becomes water-soluble at low temperatures and hardly water-soluble at high temperatures, so that the polymer material has the temperature responsiveness.

In order to block copolymerize N-isopropylacrylamide, the star polymer homopolymer synthesized as described above is dissolved in a solvent such as methanol, mixed with N-isopropylacrylamide, and polymerized by irradiation with light. You can do it. The concentration of the star homopolymer in the solution when starting the polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of N-isopropylacrylamide is preferably about 0.3 to 30% by weight. . The light irradiation conditions are preferably a light wavelength of 300 to 400 nm, an irradiation time of 1 to 30 minutes, and an irradiation intensity of about 1 μW to 10 mW / cm ² .

  The molecular weight of the block copolymer (polymer material) is preferably 3,000 to 500,000, particularly 10,000 to 100,000.

By bringing this polymer material into contact with an alkaline aqueous solution (concentration is preferably 0.1 to 4.0 N) such as NaOH, KOH, or Na ₂ CO ₃ , the homopolymer block becomes anionic by preferably about 0.5 to 120 minutes. It becomes. Preferably, the alkali treatment is performed after the polymer material is supported on the carrier.

  As the carrier, resin films such as polyvinylidene fluoride, polypropylene, polystyrene, polysulfone, polyurethane, and vinyl chloride are suitable. good.

Since the polymer material is soluble in water at a temperature lower than the predetermined temperature T, an aqueous solution having a temperature lower than the predetermined temperature may be used, and the polymer material may be supported on the support by bringing it into contact with the support and then drying. it can. The amount of the polymer material supported on the carrier surface is preferably about 0.01 mg to 1000 mg / cm ² .

  The alkali treatment is performed under a temperature condition higher than the predetermined temperature T so that the polymer material does not elute.

  The LDL adsorbent supported on the carrier is insoluble in water at a temperature higher than about 25 ° C. and is water-soluble at a temperature lower than 25 ° C.

  Therefore, LDL is removed from blood by bringing the blood and the LDL adsorbent-carrying LDL removal material into contact with each other at a temperature higher than 25 ° C., preferably at a living body temperature.

  In order to perform the LDL removal treatment of blood, a particulate LDL removal material may be packed in a column, a tube, or the like, and blood may be passed therethrough. Alternatively, the LDL removal material may be a hollow fiber, and blood may be circulated inside or outside the hollow fiber.

  It has been observed that the greater the number of branched chains of the star polymer material, the better the adhesion of the LDL adsorbent to the carrier.

  The reason for this is that the polymer chain of N-alkylacrylamide in the branched chain has an affinity for the carrier; the LDL adsorbent is attached to the carrier via the polymer chain of N-alkylacrylamide. Therefore, the more the branched chain, the better the adhesion of the LDL adsorbent to the carrier; even if one branched chain peels off from the living body, the other branched chain adheres to the carrier of the LDL adsorbent. It is conceivable that the detached branched chain is again attached to the carrier.

  The branched chain extends radially from the benzene nucleus of the star polymer material. The benzene nucleus has poor adhesion to the carrier, and the greater the number of branched chains extending in the radial direction, the more the movement in the direction away from the carrier near the benzene nucleus is suppressed. Therefore, for this reason, it is considered that the greater the number of branched chains, the better the adhesion of the LDL adsorbent to the carrier.

Example 1
i) Synthesis of iniferter According to the following reaction formula, 1,2,4,5-tetrakis (NN-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

  2.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 12.0 g of sodium N, N-diethyldithiocarbamate were added to 10 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, dried under reduced pressure, dissolved in 40 mL of chloroform, extracted with 50 mL of water, and sodium bromide was removed. After repeating this operation three times, the chloroform layer was dried with about 10 g of magnesium sulfate for 24 hours, filtered, added with n-hexane, purified by recrystallization, and a slightly light blue 1, White crystals of 2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene were obtained (yield 90%).

The measurement results of ¹ H NMR (in CDCl ₃ ) are σ7.48 (s, 2H, Ar—H), σ4.57 (s, 2H × 4, Ar—CH ₂ S—), σ4.03 (q, 2H). × 4, N—CH ₂ −, J = 10.7 Hz), σ 3.72 (q, 2H × 4, N—CH ₂ −, J = 10.7 Hz), σ 1.26 to 1.31 (t, 3H) × _8, became _{-CH 2 -CH 3, J = 6.6Hz} ).

ii) Synthesis of polymethacrylic acid homopolymer comprising 4-branched star polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis (N, N-diethyldithiocarbamate) A polymethacrylic acid homopolymer composed of a mill (polymethacrylic acid) (hereinafter sometimes referred to as pMA) was synthesized.

That is, 0.43 g of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 10 mL of toluene, and 5.2 g of methacrylic acid was added and mixed. The total amount was adjusted to 20 mL with toluene. After purging with high purity nitrogen gas for 5 minutes with vigorous stirring in a quartz cell, ultraviolet light was irradiated with a 200 W high pressure mercury lamp for 30 minutes. The irradiation intensity was adjusted to 1 mW / cm ² (250 nm) using an illuminometer (UVR-1, TOPCON, Tokyo, Japan). The polymerization solution is concentrated with an evaporator, purified by reprecipitation of the polymer with diethyl ether, dissolved in a small amount of water, filtered through a 0.2 μm filter, and lyophilized to obtain a 4-branched star homopolymer 1,2. , 4,5-Tetrakis (N, N-diethyldithiocarbamyl (polymethacrylic acid) (pMA) was obtained (40% polymerization rate), and the molecular weight was determined to be 60,000 by GPC.

iii) Synthesis of polymer material (4-branched pMA-b-pNIPAM) by block copolymerization of N-isopropylacrylamide with polymethacrylic acid homopolymer According to the following reaction formula, tetrakis (N, N-diethyl) Dithiocarbamyl (polymethacrylic acid) -block-poly- (N-isopropylacrylamide) (hereinafter sometimes referred to as pMA-b-pNIPAM) was synthesized.

  That is, 645 mg of the 4-branched pMA homopolymer synthesized in the above ii) was dissolved in 10 mL of methanol, and 1.2 g of N-isopropylacrylamide (NIPAM) was mixed to adjust the total amount to 20 mL with methanol. Photo-irradiation polymerization was performed under the same conditions as in ii), and purification was performed with a methanol / diethyl ether system to obtain a polymer material composed of a block polymer of 4-branched pMA and poly N-isopropylacrylamide (pNIPAM) (polymerization). 80%). The molecular weight was determined to be 130,000 by GPC. The ion exchange capacity was determined to be about 3.2 mEq / g by neutralization titration with 0.05 N NaOH.

iv) Immobilization to a porous carrier and anionization Polyfluoride in which water is infiltrated into micropores by immersing a polyvinylidene fluoride membrane (pore diameter 0.45 μm, film thickness 100 μm) in ethanol, followed by washing with pure water A vinylidene film was obtained. This membrane was immersed in a 0.1% solution prepared by dissolving the 4-branched pMA-b-pNIPAM synthesized in iii) in water at 20 ° C. and stirred for 60 minutes so as not to damage the membrane.

  The membrane was removed from the 4-branched pMA-b-pNIPAM solution and air dried with a dryer.

  When this 4-branched pMA-b-pNIPAM-treated polyvinylidene fluoride membrane is floated on water at 37 ° C, water is sucked into the micropores by capillary action, penetrates to the air side surface, and is confirmed to be hydrophilic by the polymethacrylic acid block It was done. It was found that the hydrophilicity was maintained even after repeated air drying and immersion in water at 37 ° C., and the 4-branched pMA-b-pNIPAM was fixed to the surface of the microporous pores of the polyvinylidene fluoride film having no reaction active sites.

  Subsequently, the pMA block was changed from H-type to Na-type by immersion in 0.05N NaOH for 10 minutes at 37 ° C., and the polyacrylic acid portion in the pMA block chain was anionic. Under such mild conditions, it could be modified to polyanionic. In addition, it wash | cleaned with the pure water of 37 degreeC after the said alkali treatment.

v) Adsorption evaluation of LDL Human fresh blood collected from a hyperlipidemic donor with a blood LDL concentration of 140 mg / dL or more was anticoagulated with 3.8% citric acid, and plasma components were collected by centrifugation. 1 mL of plasma was put into a 2 mL Eppendorf tube, and the temperature was adjusted to 37 ° C. with gentle shaking and stirring. A 45 mm ² (about 20 mg) 4-branched pMA-b-pNIPAM-treated polyvinylidene fluoride membrane was chopped into pieces of about 3 mm × 3 mm, immersed and incubated for 3 hours. After 3 hours, only the plasma was sucked up, total cholesterol, HDL, and neutral fat were measured with a final test kit, the concentration of LDL was calculated using the Friedewald equation, and LDL adsorption was performed by comparison with the original plasma concentration. The removal rate was determined (removal rate 12.2% ± 3.4%).

Comparative Example 1
The untreated polyvinylidene fluoride membrane was hydrophilized with ethanol and replaced with physiological saline. When this film was used for the same LDL removal treatment as in Example v), the LDL removal rate was 3.4% ± 0.7%.

Claims (11)

In an LDL adsorbent made of an anionic polymer material,
The LDL adsorbent characterized in that the polymer material is water-soluble at a temperature lower than a predetermined temperature (T) lower than a living body temperature and hardly water-soluble at a temperature higher than the predetermined temperature (T).   2. The LDL adsorbent according to claim 1, wherein the polymer material is a block copolymer of a homopolymer and N-alkylacrylamide, and the homopolymer block is anionic.   The LDL adsorbent according to claim 2, wherein the alkyl group of N-alkylacrylamide has 1 to 10 carbon atoms.   4. The LDL adsorbent according to claim 3, wherein the N-alkylacrylamide is N-isopropylacrylamide.   The LDL adsorbent according to any one of claims 2 to 4, wherein the polymer material is anionic by bringing the block copolymer into contact with an alkaline aqueous solution.   6. The LDL adsorbent according to claim 5, wherein the block copolymer has a molecular weight of 3,000 to 500,000.   The LDL adsorbent according to any one of claims 1 to 6, wherein the predetermined temperature (T) is a temperature between 24 to 26 ° C.   The homopolymer according to any one of claims 1 to 7, wherein the homopolymer is an iniferter having a compound having four or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule. An LDL adsorbent characterized by being a star polymer polymerized by irradiation living polymerization.   9. The compound having four or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule as defined in claim 8 having a benzene ring as a nucleus and four or more N, N- An LDL adsorbent, wherein dialkyl-dithiocarbamylmethyl molecular groups are bonded.   10. The LDL adsorbent according to claim 8, wherein the vinyl monomer is acrylic acid or methacrylic acid.   The LDL removal material by which the LDL adsorption material of any one of Claim 1 thru | or 10 is carry | supported by the support | carrier.