JP4009772B2 - Adsorbent and extracorporeal circulation column for transforming growth factor beta - Google Patents

Description

【００３４】
表１の比較例１では比表面積が大きいにも拘わらず、親水性アミノ基のリガンドがないため吸着性がない。比較例２は親水性でないアミノ基のリガンドのため吸着能が無い。比較例３からスルホン酸基のリガンドは吸着性がないことが分かる。また、比較例４は親水性アミノ基のリガンドであるが、実施例３及び実施例４と異なり比表面積が低いため吸着能が殆ど無い。比較例５及び比較例６は比表面積は小さくないが、親水性アミノ基のリガンドがないため吸着能がない。これらの結果から、吸着には親水性アミンの官能基が必要であること、比表面積が０．０４平方メートルでは吸着能が低いことが分かる。また、親水性アミンの中でも、窒素原子１個当たりの炭素数が１４のものより１０および８のものの吸着能が大きいことが分かる。
【００３５】
【発明の効果】
本発明により、免疫抑制性物質を効率よく吸着除去することが可能となり、癌の患者の治療に有用である。[0001]
BACKGROUND OF THE INVENTION
The present invention removes TGFβ in which other proteins are bound to TGFβ having a molecular weight of about 25,000, which is an immunosuppressive protein that promotes the formation of cancer if it is excessively present in the blood. The present invention relates to an adsorbent and an extracorporeal circulation column.
[0002]
[Prior art]
Even today, with the development of medicine, cancer is still one of the main causes of death in Japan. The cause is that there are cancer cells that cannot be removed by surgery in patients, and anticancer drug treatment and radiation therapy have been performed to remove them. However, since these also damage normal cells, cancer cells cannot be completely removed while maintaining the patient's life. On the other hand, recently, cell therapy has been actively tried to improve the immunity of patients and eliminate cancer with their own white blood cells. The most promising is dendritic cell infusion therapy in which a patient's dendritic cells are stimulated with cancer antigens outside the body and then returned to the patient to induce and treat cancer-specific killer cells (CTLs). However, in many cases, CTL can be induced from the blood of healthy animals, but cannot be induced from cancer-bearing animals at the end of cancer. In animal experiments, even if good results are obtained, there is almost no visible effect in clinical practice. This may be because a substance that suppresses immunity exists in the blood of the patient. Representative examples of such immunosuppressive substances are transforming growth factor beta (TGFβ) and immunosuppressive acidic proteins. This substance is thought to inhibit CTL induction and functional expression. TGFβ is an important substance that regulates immunity with a protein that is also present in the blood of a healthy person, but is thought to increase abnormally as cancer progresses and to help cancer cells grow. TGFβ alone is a protein having a molecular weight of about 25,000. However, TGFβ is a substance that is difficult to adsorb and remove with conventional adsorbents because it binds to other proteins in blood and has a molecular weight of around 100,000. Therefore, no adsorbent is known that can efficiently remove abnormally increased TGFβ from the blood of cancer patients. Immunosuppressive acidic protein is a protein having a molecular weight of about 50,000 and is clinically used as a marker of cancer malignancy, but is said to suppress immunity. Attempts were made to remove it with an activated carbon column, but the therapeutic effect was not clear due to insufficient adsorption capacity. In addition, activated carbon is easy to produce powder, so it is not suitable for use in direct contact with blood. The removal of these immunosuppressive substances is theoretically effective for plasma exchange, but has the inherent disadvantage that the risk of infection is unavoidable.
[0003]
[Problems to be solved by the invention]
The present invention is generally ubiquitous in view of the problems of the prior art, and can select TGFβ obtained by binding other proteins directly to TGFβ having a molecular weight of about 25,000 in blood directly from a body fluid with high efficiency. It is intended to provide an adsorbent for TGFβ, which can be adsorbed on the surface and can be safely circulated outside the body, and in which TGFβ having a molecular weight of about 25,000 in blood is bound to other proteins, and thus useful for cancer treatment.
[0004]
[Means for Solving the Problems]
That is, the present invention has a specific surface area of more than 0.1 square meters per gram, formed by bonding a hydrophilic amine residue to an insoluble carrier, TGF [beta other protein molecular weight of about 25000 TGF [beta in the blood is bound It is an adsorbent for an immunosuppressive protein characterized by adsorbing.
[0005]
Moreover, this invention is an extracorporeal circulation column filled with said adsorption material.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Subsequently, the present invention will be described in more detail.
[0007]
The water-insoluble carrier used in the present invention is not particularly limited as long as it is insoluble in water and can immobilize a hydrophilic amine.
Specific examples of the water-insoluble carrier include poly (aromatic vinyl compounds) represented by polystyrene, poly (p-phenylene ether sulfone) and-{(p-C ₆ H ₄ ) -C (CH ₃ ) _2- (p _{-C 6 H 4) -O- (p} -C 6 H 4) -SO 2 - (p-C 6 H 4) -O-} n - ( hereinafter abbreviated as U del polysulfone) polysulfone represented by like Examples thereof include polymers, polyetherimides, polyimides, polyamides, polyethers, polyphenylene sulfides, and the like that can immobilize hydrophilic amines. Reactive functional groups for immobilizing hydrophilic amines include active halogen groups such as halomethyl, haloacetyl, haloacetamidomethyl, and halogenated alkyl groups, epoxide groups, carboxyl groups, isocyanate groups, and thioisocyanate Groups, acid anhydride groups, and the like. In particular, active halogen groups, especially haloacetyl groups, are easy to produce, have moderately high reactivity, and have a mild hydrophilic amine immobilization reaction. It is preferable because it can be carried out under conditions and the covalent bond formed at this time is chemically stable. More specific examples include chloroacetamidomethyl polystyrene, chloracetamidomethylated Udel polysulfone, chloroacetamidomethylated polyetherimide, and the like. Furthermore, when these polymers are soluble in an organic solvent, there is an advantage that they are easy to mold.
[0008]
The hydrophilic amine residue as used in the field of this invention means the thing of the state which the amine which melt | dissolves in water alone or dissolves water chemically couple | bonded with the polymer. Furthermore, as the hydrophilic amine of the hydrophilic amine residue, in terms of the number of carbon atoms, one having 18 or less carbon atoms per nitrogen atom corresponds to this.
[0009]
Furthermore, among hydrophilic amines, those bonded with a quaternary ammonium group obtained from a tertiary amine having an alkyl group having 3 to 18 carbon atoms, particularly 4 to 14 carbon atoms per nitrogen atom are excellent. . Specific examples of such tertiary amines include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl-hexyl. Examples include amines. Further, those containing a hydroxyl group or ether group in which the alkyl group constituting the hydrophilic amine is a hydrophilic group, such as N, N-dimethyl-6-hydroxyhexylamine and N, N-dimethyl-4-methoxybutylamine are also included. It can be preferably used as a hydrophilic amine.
[0010]
The bonding density of hydrophilic amine residues in the present invention varies depending on the chemical structure and use of the water-insoluble polymer. However, if the amount is too small, the function tends not to be expressed. Since the physical strength of the coalescence deteriorates and the function as an adsorbent tends to decrease, the density is 0.01 to 2.0 mol, more preferably 0.1 to 1. mol per repeating unit of the water-insoluble polymer. 0 mole is good.
[0011]
It is important that the surface area of the adsorbent of the present invention is 0.1 square meter or more per gram of adsorbent, more preferably 1 square meter or more. However, since it cannot be infinitely large, there is a practical limit, and 100 square meters or less is preferable. This surface area can be determined by a nitrogen gas adsorption method (BET method).
[0012]
The adsorbent of the present invention is obtained by molding a water-insoluble polymer bonded with a hydrophilic amine residue into a film, a fiber, a granular material or an assembly thereof, or a water-insoluble polymer bonded with a hydrophilic amine residue. It can be obtained by coating a substrate of any one of a membrane, a fiber and a granular material, or by bonding a hydrophilic amine to a molded product such as a membrane or fiber of a water-insoluble polymer.
[0013]
For the production of a water-insoluble carrier molded product bonded with a hydrophilic amine residue, a heterogeneous reaction method in which a hydrophilic amine solution is brought into contact with a water-insoluble polymer molded product, and the water-insoluble polymer solution and the hydrophilic There is a homogeneous reaction method in which a solution of a reactive amine is mixed and reacted and then molded. As an example of a heterogeneous reaction method, a molded product such as a fiber or hollow fiber of chloroacetamidomethylated polysulfone is immersed in an isopropanol solution such as dimethylhexylamine or polyalkyleneimine and reacted at a temperature of 0 to 100 ° C. This is easily achieved. An example of the method by homogeneous reaction is achieved by adding the corresponding polyamine to a solution of chloroacetamidomethylated polysulfone and reacting at a temperature of 0 to 100 ° C. The amount is not particularly limited, but it is desirable to use at least 1 mole per mole of haloacetamidomethyl groups to obtain a soluble polymer. In particular, in the case of polyamine, it is preferable to use a large excess of hydrophilic amine in order to obtain a soluble polymer.
[0014]
As the reaction solvent, a solvent having a high polarity such as water, methanol, ethanol, isopropanol, dimethyl sulfoxide, N, N-dimethylformamide (DMF) has an advantage that the reaction proceeds faster. The heterogeneous reaction is not particularly limited as long as it is a solvent in which the hydrophilic amine is soluble. When the reaction is carried out in a homogeneous system, a solvent in which both the water-insoluble carrier and the hydrophilic amine are dissolved, specifically tetrahydrofuran, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone Etc. are preferably used. In addition, a method of surface-treating a molded product is also possible, and for this purpose, a solvent that dissolves a hydrophilic amine without dissolving polysulfone, such as water, methanol, and ethanol, is preferably used.
[0015]
When the adsorbent polymer of the present invention is coated on the surface of a molded product such as polyester fiber, nylon fiber, polyphenylene sulfide fiber, etc., there is an advantage that a high-order molded product having a large surface area can be obtained easily and inexpensively. Coating can be easily achieved by immersing a nylon knitted fabric or woven fabric in the adsorbent of the present invention dissolved in a low-boiling solvent such as methylene chloride or tetrahydrofuran and evaporating the solvent. Further, a wet coating method in which a solution dissolved in a solvent such as N, N-dimethylformamide is put into a poor solvent for a polymer such as water can also be used. The polymer of the molded article to be coated may be any polyamide, polyurethane, polyimide, polysulfone, polyvinyl chloride, polyester, etc., as long as it has good adhesion to the adsorbent polymer of the present invention, and there is no particular limitation on the type. An amide polymer such as nylon or polyetherimide is preferably used because of its particularly good adhesion.
[0016]
In the above molding and coating on the base material, it is also preferable to use a hollow fiber as a fiber to be employed in the form of the molded product or base material. In this case, since an adsorbent having a filtration function can be produced, there is an advantage that an immunosuppressive substance can be removed while being used as an artificial dialyzer or a plasma separator.
[0017]
Adsorbent of the present invention for the purpose of removal of TGF [beta bound by other proteins in the molecular weight of about 25000 TGF [beta in the blood, while packed in a column, employed in extracorporeal circulation treatment of cancer such diseases. This column can be produced by packing the adsorbent of the present invention in the form of cotton, cylinder, or felt into a cylindrical column having an appropriate size so that the void volume is about 200 mL or less. The adsorbent of the present invention can also be used for the purpose of removing immunosuppressive proteins from blood for transfusion, serum, and plasma.
[0018]
【Example】
Hereinafter, the present invention will be described more specifically by experimental examples.
[0019]
In addition, the evaluation method in a present Example followed the following.
1. Analysis of components in blood TGFβ1 concentration was determined using a human TGF-β1 immunoassay kit from Zenzyme Techne. The concentration of the immunosuppressive acidic protein was determined using a rat IAP plate manufactured by Sanko Junyaku Co., Ltd. The albumin concentration was determined by albumin B-Test Wako, which is an albumin analysis kit.
[0020]
(Water-insoluble carrier)
Thirty-six sea-island composite fibers, each of which is made of a core-sheath composite, were obtained using the following components under the spinning conditions of a spinning speed of 800 m / min and a draw ratio of 3 times.
Island core component; Polypropylene island sheath component: 90% polystyrene, 10% polypropylene
Sea component; Polyethylene terephthalate composite ratio obtained by copolymerization of 3% of 5-sodium sulfoisophthalic acid; Core: sheath: sea = 40: 40: 20.
This sea component was dissolved in a hot caustic soda aqueous solution to obtain a raw yarn 1 having a diameter of 4 μm as a core-sheath type polypropylene reinforced polystyrene fiber.
[0021]
Further, the discharge amount and the draw ratio were appropriately changed, and similarly, a raw yarn 2 having a diameter of 10 μm and a raw yarn 3 having a diameter of 50 μm were obtained.
[0022]
(Intermediate)
After 3 g of paraformaldehyde was dissolved at 20 ° C. in a mixed solution of 600 mL of nitrobenzene and 390 mL of sulfuric acid, the solution was cooled to 0 ° C., and 75.9 g of N-methylol-α-chloroacetamide was added and dissolved at 5 ° C. or lower. 10 g of the original yarn 1 prepared above was soaked in this, and allowed to stand at room temperature for 2 hours. Thereafter, the fiber was taken out and placed in a large excess of cold methanol for washing. The fiber was thoroughly washed with methanol, washed with water, and dried to obtain 15.0 g of α-chloroacetamidomethylated polystyrene fiber (intermediate 1). Intermediate 1 was also used as Comparative Example 1. Further, 10 g of raw yarn 2 and 10 g of raw yarn 3 were treated in the same manner to obtain Intermediate 2 (yield 14.4 g) and Intermediate 3 (yield 12.5 g).
[0023]
(Fixation by heterogeneous reaction of hydrophilic amine)
5 g of Intermediate 1 was immersed in a solution of 50 g of N, N-dimethylhexylamine and 8 g of potassium iodide in 360 mL of DMF, and heated in a 85 ° C. bath for 3 hours. The fiber was immersed in a 1 mol / L saline solution, then washed with water and vacuum dried to obtain 7.3 g of dimethylhexylammonium-modified fiber (Example 1).
[0024]
Further, 5 g of Intermediate 1 was immersed in a solution of 50 g of N, N-dimethyloctylamine and 8 g of potassium iodide in 360 mL of DMF, and heated in a bath at 85 ° C. for 3 hours. The fiber was washed with isopropanol, immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 8.3 g of dimethyloctylammonium-containing fiber (Example 2).
[0025]
Further, 5 g of Intermediate 1 was immersed in a solution of 50 g of N, N-dimethyllaurylamine and 8 g of potassium iodide in 360 mL of DMF, and heated in a 85 ° C. bath for 3 hours. The fiber was washed with isopropanol, immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 9.3 g of dimethyllauryl ammoniumated fiber (Example 3). Moreover, the intermediate body 2 and the intermediate body 3 were processed similarly, and Example 4 (specific surface area 1.4m < ² > / g) and the comparative example 4 (specific surface area 0.04m < ² > / g) were obtained, respectively.
[0026]
(Immobilization of non-hydrophilic amine by heterogeneous reaction)
Further, as a non-hydrophilic amine, 5 g of the intermediate 1 was immersed in a solution of 50 g of stearylamine in 360 mL of ethanol, and heated in a bath at 85 ° C. for 3 hours. The fiber was washed with isopropanol, washed with water, and dried under vacuum to obtain 7.2 g of stearyl aminated fiber (Comparative Example 2).
[0027]
(Sulfonated fiber)
In addition, 5 g of the original yarn 1 is immersed in 50 mL of sulfuric acid in which 500 mg of paraformaldehyde is dissolved, heated at 95 ° C. for 1 hour, washed with water, washed with 1 mol / L saline solution, washed with water, and dried successively. 7.3 g of sulfonated fiber (Comparative Example 3) was obtained.
[0028]
(Synthesis and coating of hydrophilic amine bond polymer)
After cooling a mixed solution of 16 mL of nitrobenzene and 32 mL of sulfuric acid to 0 ° C., 4.2 g of N-methylol-α-chloroacetamide was added and dissolved, and this was dissolved in a 3 L nitrobenzene solution of Eudelpolysulfone P3500 at 10 ° C. 300 g / 3 L) with good stirring. Furthermore, it 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, dried, and reprecipitated from dimethylformamide / methanol to obtain 303 g of α-chloroacetamidomethylated polysulfone (substitution rate: 0.05; polymer C).
[0029]
A solution obtained by dissolving 60 g of polyethyleneimine (average molecular weight 10,000: Wako Pure Chemical Industries, Ltd.) in 300 mL of DMF and 300 mL of DMF solution containing 30 g of Polymer C were mixed and stirred at room temperature for 48 hours. The reaction mixture was added to a large excess of saturated brine, and the precipitated polymer was collected by filtration. The polymer was washed with water, dried, and reprecipitated from dimethylformamide / methanol to prepare 27 g of N-alkylated polyalkylenimine-immobilized polysulfone (Polymer D).
[0030]
20 g of polyethylene terephthalate fiber having a single yarn diameter of 3.5 μm is dipped in 250 mL of methylene chloride containing 5 g of the above polymer D. After 20 hours, the cotton is taken out, drained and air-dried, and 21 g of coated cotton. (Example 5) was obtained.
[0031]
(Nonionic functional group adsorbent)
Dissolve 5 g of cellulose acetate in 250 ml of methylene chloride, soak 20 g of polyethylene terephthalate fiber with a single yarn diameter of 3.5 μm, take out the cotton after 20 hours, drain the solution, air dry, and 21 g of coated cotton (Comparative Example 5 )
[0032]
(Evaluation of adsorption capacity)
Blood was collected from 5 cancer-bearing rats prepared by inoculating 2 × 10 ⁸ YS cells (provided by Tohoku University Aging Research Institute) subcutaneously in the back of 8-week-old HOS: Donryu rats (male). Prepared. 50 mL of an adsorbent was added to 1 mL of the serum and shaken at 37 ° C. for 4 hours. Each protein concentration in the supernatant was measured, and the results shown in Table 1 were obtained.
[0033]
[Table 1]

[0034]
Although Comparative Example 1 in Table 1 has a large specific surface area, it has no adsorptivity because there is no hydrophilic amino group ligand. Comparative Example 2 has no adsorptive capacity due to a non-hydrophilic amino group ligand. It can be seen from Comparative Example 3 that the sulfonic acid group ligand has no adsorptivity. Comparative Example 4 is a hydrophilic amino group ligand, but unlike Examples 3 and 4, it has almost no adsorptive capacity because of its low specific surface area. In Comparative Examples 5 and 6, the specific surface area is not small, but there is no adsorption ability because there is no hydrophilic amino group ligand. From these results, it is understood that a functional group of hydrophilic amine is necessary for adsorption, and that the adsorption capacity is low when the specific surface area is 0.04 square meters. It can also be seen that among hydrophilic amines, those having 10 and 8 carbon atoms per nitrogen atom have a higher adsorption capacity than 14 carbon atoms.
[0035]
【The invention's effect】
According to the present invention, an immunosuppressive substance can be efficiently adsorbed and removed, which is useful for the treatment of cancer patients.

Claims (8)

The specific surface area is 0.1 square meter or more per gram, and a molecular weight of about 25,000 is formed by binding a quaternary ammonium group having 3 to 18 carbon atoms per nitrogen atom to a water-insoluble support. An adsorbent for transforming growth factor beta, in which other proteins are bound to transforming growth factor beta. Transforming Growth Growth Factor beta adsorbent claim 1 molecular weight 25,000 about Transforming Growth Growth Factor Beta other proteins described bonded water-insoluble carrier is a polysulfone-based polymer. Water-insoluble polymer is poly (aromatic vinyl compound) in a claim 1 Transforming Growth Growth Factor beta adsorbent molecular weight other protein Transforming Growth Growth Factor Beta of about 25,000 is bonded according . Quaternary ammonium groups bound water insoluble polymer membrane nitrogen per atom carbon number of 3 to 18 or less, fibers, granules or according to any one of claims 1 to 3 was molded into these assembly An adsorbent for transforming growth factor beta, in which other proteins are bound to transforming growth factor beta having a molecular weight of about 25,000. Quaternary water-insoluble polymer an ammonium group bonded at most one nitrogen atom per carbon number of 3 to 18, and film, fiber, claim 1-3 which allowed coated on either surface of the granules An adsorbent for transforming growth factor beta, in which another protein is bound to transforming growth factor beta having a molecular weight of about 25,000. It is characterized in that the adsorbing ability to transforming growth factor beta in which other proteins are bound to transforming growth factor beta having a molecular weight of about 25,000 in blood is 340 mg or more per gram of adsorbent. An adsorbent for transforming growth factor beta obtained by binding another protein to the transforming growth factor beta having a molecular weight of about 25,000 according to any one of claims 1 to 5 . An extracorporeal circulation column packed with a transforming growth factor beta adsorbent in which another protein is bound to the transforming growth factor beta having a molecular weight of about 25,000 according to any one of claims 1 to 6 . An extracorporeal circulation for cancer treatment, which is filled with an adsorbent of transforming growth factor beta in which another protein is bound to transforming growth factor beta having a molecular weight of about 25,000 according to any one of claims 1 to 6. column.