JP2003111834A - Adsorbent for immunosuppressive protein and column for extracorporeal circulation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunosuppressive protein adsorbent that can gain widespread use for directly and selectively adsorbing immunosuppressive substances such as TGFβ and immunosuppressive acidic proteins from humor with high efficiency and that allows extracorporeal circulation with safety to serve to treat cancers. SOLUTION: The adsorbent for immunosuppressive substances has a specific surface area of 0.1 square meters per gram and contains a hydrophilic amine residual group coupled to a water-insoluble carrier. The column for extracorporeal circulation is filled with the adsorbent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention is for removing immunosuppressive proteins such as TGFβ and immunosuppressive acidic proteins which, if excessively present in blood, lead to a significant decrease in immunity and promote the formation of cancer. Adsorbent and extracorporeal circulation column.

[0002]

2. Description of the Related Art Even with the advance of medical science, cancer remains one of the leading causes of death in the Japanese people. The reason is that the patient has cancer cells that cannot be removed by surgery,
Anticancer drug treatment and radiation treatment have been performed to remove them. However, since these also damage normal cells, the cancer cells cannot be completely removed while maintaining the life of the patient. On the other hand, recently, cell therapy has been actively attempted to enhance the immunity of the patient and eliminate the cancer with the patient's own white blood cells. The most promising one is dendritic cell infusion therapy in which a patient's dendritic cells are stimulated with cancer antigen in vitro and then returned to the patient to induce and treat cancer-specific killer cells (CTL). However, CT from the blood of healthy animals
Even if L can be induced, it often cannot be induced from a tumor-bearing animal at the end stage of cancer. Moreover, even if good results are obtained in animal experiments, in most cases clinically no observable effects are obtained. It is considered that this is because there are substances that suppress immunity in the blood of patients. Typical examples of the immunosuppressive substances are transforming growth factor beta (TGFβ) and immunosuppressive acidic protein. This substance is C
It is considered that TL induction and functional expression are inhibited. T
GFβ is a protein that is also present in the blood of healthy people and is an important substance that regulates the immune action, but it is thought to increase abnormally with the progression of cancer and help the growth of cancer cells.
TGFβ is a protein having a molecular weight of about 25,000 by itself, but since it is bound to other proteins in blood and has a molecular weight of about 100,000, it is a substance difficult to adsorb and remove with a conventional adsorbent. Therefore, an adsorbent that can efficiently remove the abnormally increased TGFβ from the blood of cancer patients is not known. Further, the immunosuppressive acidic protein is a protein having a molecular weight of about 50,000 and is clinically used as a marker of malignancy of cancer, but is said to suppress immunity. An attempt was made to remove it using an activated carbon column, but the therapeutic effect was not clear, probably because the adsorption capacity was insufficient. In addition, activated carbon is not suitable for applications where it comes into direct contact with blood because activated carbon easily produces powder. In theory, plasma exchange is also effective in removing these immunosuppressive substances, but there is an essential drawback that the risk of infection is unavoidable.

[0003]

In view of the above problems of the prior art, the present invention is widely applicable, and it is possible to directly obtain an immunosuppressive substance such as TGFβ or immunosuppressive acidic protein with high efficiency from a body fluid. It is an object of the present invention to provide an immunosuppressive protein adsorbent that can be selectively adsorbed by and can be safely extracorporeally circulated, and eventually be useful for treating cancer.

[0004]

That is, the present invention has a specific surface area of 0.1 square meter or more per gram,
An adsorbent for an immunosuppressive protein, which comprises a hydrophilic amine residue bound to an insoluble carrier.

Further, the present invention is an extracorporeal circulation column packed with the above-mentioned adsorbent.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail.

The water-insoluble carrier used in the present invention is water.
So long as it is insoluble in and can immobilize hydrophilic amines
There is no particular limitation. Specific examples of the water-insoluble carrier include po
Poly (aromatic vinyl compound) represented by styrene,
Poly (p-phenylene ether sulfone) or-{(p-
C₆H_Four) -C (CH₃)₂-(P-C₆H_Four) -O-
(P-C₆H_Four) -SO₂-(P-C₆H _Four) -O-}
_n-(Uder polysulfone)
Represented polysulfone polymer, polyetherimide,
Polyimide, polyamide, polyether, polyphenylene
And immobilize hydrophilic amine
I can give you what I can. To immobilize hydrophilic amines
The reactive functional groups of are halomethyl group, haloacetyl group
Group, haloacetamidomethyl group, halogenated alkyl group
Active halogen groups such as, epoxide groups, carboxyl
Group, isocyanic acid group, thioisocyanic acid group, acid anhydride group
And the like, but especially active halogen
Group, especially haloacetyl group, is easy to manufacture and
The reactivity is moderately high, and the immobilization reaction of hydrophilic amine is mild.
It can be carried out under conditions, and the covalent bond generated at this time is chemically
Is stable, which is preferable. As a more specific example,
Chloracetamidomethyl polystyrene, chloracetate
Amdelmethylated Udel Polysulfone, Chlorua
Cetamide methylated polyether imide etc.
To be Furthermore, these polymers are compatible with organic solvents.
Melting has the advantage of being easy to mold.

The term "hydrophilic amine residue" as used in the present invention means a state in which a water-soluble or water-soluble amine is chemically bonded to a polymer. Further, the hydrophilic amine of the hydrophilic amine residue corresponds to one having a carbon number of 18 or less per nitrogen atom in terms of carbon number.

Further, among the hydrophilic amines, the number of carbon atoms per nitrogen atom is 3 or more and 18 or less, and particularly 4 or more and 1 or less.
Those having a quaternary ammonium group obtained from a tertiary amine having an alkyl group of 4 or less are excellent. Specific examples of such a tertiary amine include trimethylamine, triethylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, N, N-dimethyllaurylamine, N-methyl-N-ethyl-hexyl. Examples include amines. Further, those in which the alkyl group constituting the hydrophilic amine contains a hydroxyl group or an ether group, which are hydrophilic groups, such as N, N-dimethyl-6-hydroxyhexylamine and N, N-dimethyl-4-methoxybutylamine It can be preferably used as a hydrophilic amine.

The bond density of the hydrophilic amine residue in the present invention varies depending on the chemical structure and use of the water-insoluble polymer, but if it is too small, its function tends not to be exhibited, while if it is too large, it is immobilized. Since the physical strength of the polymer afterwards tends to be poor and the function as an adsorbent tends to be lowered, the density is 0. 0 per repeating unit of the water-insoluble polymer.
The amount is preferably 01 to 2.0 mol, more preferably 0.1 to 1.0 mol.

It is important that the surface area of the adsorbent of the present invention is 0.1 m 2 or more per gram of the adsorbent, and more preferably 1 m 2 or more. However, since it cannot be made infinitely large, there is a practical limit,
100 square meters or less is preferable. This surface area can be determined by the nitrogen gas adsorption method (BET method).

The adsorbent of the present invention is formed by molding a water-insoluble polymer having a hydrophilic amine residue bonded into a film, a fiber, a granular material or an assembly thereof, or a water-insoluble polymer having a hydrophilic amine residue bonded. It can be obtained by coating the polymer with a base material of a film, a fiber, or a granular material, or by bonding a hydrophilic amine to a molded product such as a water-insoluble polymer film or a fiber.

In order to produce a molded product of a water-insoluble carrier having a hydrophilic amine residue bonded thereto, a method of heterogeneous reaction in which a solution of a hydrophilic amine is brought into contact with a molded product of a water-insoluble polymer, There is a homogeneous reaction method in which a solution and a hydrophilic amine solution are mixed and reacted, and then molded. As an example of the heterogeneous reaction method, a molded article such as a chloroacetamidomethylated polysulfone fiber or hollow fiber is dipped 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 homogeneous reaction method is as follows. Add the corresponding polyamine to a solution of chloroacetamidomethylated polysulfone, and add 0
This is achieved by reacting at a temperature of ~ 100 ° C. The amount is not particularly limited, but in order to obtain a soluble polymer, it is desirable to use 1 times or more moles with respect to the haloacetamidomethyl group. 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.

As the reaction solvent, a solvent having a high polarity such as water, methanol, ethanol, isopropanol, dimethylsulfoxide, N, N-dimethylformamide (DMF) has the 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 can be dissolved. 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, dimethylsulfoxide,
N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone and the like are preferably used. A method of surface-treating the molded product is also possible, and for that purpose, a solvent such as water, methanol or ethanol that dissolves the hydrophilic amine without dissolving the polysulfone is preferably used.

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, there is an advantage that a high-order molded product having a large surface area can be obtained easily and inexpensively.
The coating can be easily accomplished by immersing the nylon knitted fabric or woven fabric in a solution of the adsorbent of the present invention in a low boiling point solvent such as methylene chloride or tetrahydrofuran, and then evaporating the solvent. Further, a wet coating method can also be used in which a substance dissolved in a solvent such as N, N-dimethylformamide is put in a poor solvent for a polymer such as water. The polymer of the molded product to be coated may be any polymer having good adhesiveness to the adsorbent polymer of the present invention, such as polyamide, polyurethane, polyimide, polysulfone, polyvinyl chloride, polyester, etc., but the type thereof is not particularly limited. Amide polymers such as nylon, polyetherimide and the like have particularly good adhesiveness and are preferably used.

In the above-mentioned molding or coating on the base material, it is also preferable to use hollow fibers as the fibers employed in the form of the molded product or the base material. In this case, since an adsorbent having a filtering function can be prepared, there is an advantage that the immunosuppressive substance can be removed while being used as an artificial dialyzer or a plasma separator.

The adsorbent of the present invention is used for the purpose of removing TGFβ, immunosuppressive acidic proteins and other immunosuppressive proteins in blood,
It is used for extracorporeal circulation treatment of diseases such as cancer in a state packed in a column. The present column can be prepared by filling the adsorbent of the present invention in the form of cotton, cylinder knit, or felt into a cylindrical column of appropriate size such 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]

EXAMPLES The present invention will be described in more detail with reference to experimental examples.

The evaluation method in this example was as follows. 1. Analysis of components in blood The concentration of TGFβ1 is the human TGF- of Zenzyme Techne.
Determined using the β1 immunoassay kit. The concentration of immunosuppressive acidic protein was determined using a rat IAP plate manufactured by Sanko Junyaku Co., Ltd. The albumin concentration was determined using an albumin analysis kit, Albumin B-Test Wako.

(Water-insoluble carrier) A sea-island composite fiber having 36 islands, wherein the island is further composed of a core-sheath composite, was obtained using the following components under the spinning conditions of a spinning speed of 800 m / min and a draw ratio of 3 times. It was Island core component: Polypropylene Island sheath component: Polystyrene 90%, Polypropylene 10
% Sea component; polyethylene terephthalate composite ratio obtained by copolymerizing 5% sodium sulfoisophthalic acid 3%; core: sheath: sea = 40:40:20. This sea component is dissolved in a hot caustic soda aqueous solution to form a core-sheath type polypropylene reinforced polystyrene fiber having a diameter of 4 μm.
Yarn 1 was obtained.

Further, the discharge amount and the draw ratio are appropriately changed, and similarly, a yarn 2 having a diameter of 10 μm and a yarn 3 having a diameter of 50 μm are obtained.
Got

(Intermediate) 3 g of paraformaldehyde was dissolved in a mixed solution of 600 mL of nitrobenzene and 390 mL of sulfuric acid at 20 ° C., then 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 yarn 1 prepared above was dipped in this, and allowed to stand at room temperature for 2 hours. Then, the fiber was taken out, put into a large excess of cold methanol, and washed. After thoroughly washing the fibers with methanol, washing with water and drying, 15.
0 g of α-chloroacetamide methylated polystyrene fiber (intermediate 1) was obtained. Intermediate 1 was also used as Comparative Example 1. Further, 10 g of the raw yarn 2 and 10 g of the raw yarn 3 were treated in the same manner to obtain Intermediate 2 (yield 14.4 g) and Intermediate 3 (yield 12.5 g).

(Immobilization of Hydrophilic Amine by Heterogeneous Reaction) 5 g of a solution prepared by dissolving 50 g of N, N-dimethylhexylamine and 8 g of potassium iodide in 360 mL of DMF.
Intermediate 1 was immersed and heated in a bath at 85 ° C. for 3 hours.
After immersing the fiber in 1 mol / L salt solution, washing it with water,
It was vacuum dried to obtain 7.3 g of dimethylhexylammonium fiber (Example 1).

Further, N, N-dimethyloctylamine 5
Immerse 5 g of Intermediate 1 in a solution of 0 g and 8 g of potassium iodide in 360 mL of DMF.
Heated for hours. The fiber was washed with isopropanol, immersed in a 1 mol / L concentration saline solution, washed with water, and vacuum dried to obtain 8.3 g of a dimethyloctylammonium-modified fiber (Example 2).

Further, N, N-dimethyllaurylamine 5
Immerse 5 g of Intermediate 1 in a solution of 0 g and 8 g of potassium iodide in 360 mL of DMF.
Heated for hours. The fiber was washed with isopropanol, immersed in a salt solution of 1 mol / L concentration, washed with water, and vacuum dried to obtain 9.3 g of dimethyllauryl ammonium modified fiber (Example 3). Moreover, the intermediate body 2 and the intermediate body 3 were treated in the same manner to obtain Example 4 (specific surface area 1.4 m).
² / g) and Comparative Example 4 (specific surface area 0.04 m ² / g) were obtained.

(Immobilization of non-hydrophilic amine by heterogeneous reaction) Further, as non-hydrophilic amine, stearylamine 5
5 g of Intermediate 1 was immersed in a solution of 0 g 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 vacuum dried to obtain 7.2 g of stearyl aminated fiber (Comparative Example 2).

(Sulfonated fiber) Further, 5 g of the raw yarn 1 was immersed in 50 mL of sulfuric acid in which 500 mg of paraformaldehyde was dissolved, heated at 95 ° C. for 1 hour, and then washed with water and washed with a 1 mol / L concentration saline solution. , Wash with water and dry sequentially,
7.3 g of sulfonated fiber (Comparative Example 3) was obtained.

(Synthesis and coating of hydrophilic amine-bonded 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 mixed with 10
It was added to a 3 L nitrobenzene solution (300 g / 3 L) of Udel Polysulfone P3500 at 0 ° C. with good stirring. Furthermore, it stirred at room temperature for 3 hours. Then, the reaction mixture was put into a large excess of cold methanol to precipitate the polymer. The precipitate was thoroughly washed with methanol, dried, and then reprecipitated from dimethylformamide / methanol to obtain 303 g of α-chloroacetamidomethylated polysulfone (substitution rate: 0.05; polymer C).

Polyethyleneimine (average molecular weight 1000
(0: Wako Pure Chemical Industries, Ltd.) 60 g of a solution prepared by dissolving it in 300 mL of DMF was mixed with a 300 mL DMF solution containing 30 g of Polymer C, and the mixture was stirred at room temperature for 48 hours. The reaction mixture was added to a large excess of saturated saline, and the precipitated polymer was collected by filtration.
The polymer was washed with water, dried, and then reprecipitated from dimethylformamide / methanol to prepare 27 g of N-alkylated polyalkyleneimine-immobilized polysulfone (Polymer D).

Methylene chloride 25 containing 5 g of the above polymer D
20 g of polyethylene terephthalate fiber having a diameter of 3.5 μm of a single yarn was dipped in 0 mL of the solution, and after 20 hours, the cotton was taken out, drained and air dried to obtain 21 g of coated cotton (Example 5).

(Nonionic functional group adsorbent) 5 g of cellulose acetate was dissolved in 250 ml of methylene chloride, 20 g of polyethylene terephthalate fiber having a single yarn diameter of 3.5 μm was soaked, and after 20 hours, the cotton was taken out and the liquid was removed. It was cut and air-dried to obtain 21 g of coated cotton (Comparative Example 5).

(Evaluation of Adsorption Ability) HOS: Don for 8 weeks
Blood was collected from 5 tumor-bearing rats prepared by inoculating 2 × 10 ⁸ YS cells (provided by Tohoku University Aging Research Institute) subcutaneously on the back of ryu rat (male) to prepare 30 mL of serum. 50 mg of an adsorbent was added to 1 mL of the serum and shaken at 37 ° C. for 4 hours. The concentration of each protein in the supernatant was measured, and the results shown in Table 1 were obtained.

[0033]

[Table 1]

In Comparative Example 1 of Table 1, despite having a large specific surface area, there is no adsorptive property because there is no hydrophilic amino group ligand. Comparative Example 2 has no adsorptive ability because it is a non-hydrophilic amino group ligand. From Comparative Example 3, it can be seen that the ligand having a sulfonic acid group has no adsorptivity. In addition, Comparative Example 4 is a hydrophilic amino group ligand, but Examples 3 and 4
Unlike that, it has almost no adsorption capacity because of its low specific surface area. Comparative Examples 5 and 6 are not small in specific surface area, but have no adsorptive ability because there is no hydrophilic amino group ligand. From these results, it is understood that the adsorption requires a functional group of hydrophilic amine, and that the adsorption capacity is low when the specific surface area is 0.04 square meters. Further, among the hydrophilic amines, it can be seen that the adsorption ability of 10 and 8 having a carbon number per one nitrogen atom is larger than that having 14 carbon atoms.

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, an immunosuppressive substance can be efficiently adsorbed and removed, which is useful for the treatment of cancer patients.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) B01J 20/28 B01J 20/28 AZ (72) Inventor Koji Watanabe 1-1-1, Sonoyama, Otsu City, Shiga Prefecture No. Toray Co., Ltd. Shiga Plant F term (reference) 4C077 AA12 BB03 CC06 EE01 HH03 JJ03 KK11 LL02 MM03 NN04 PP08 PP12 PP14 PP15 4G066 AC14C AC31C AD06B AD10B BA03 BA09 CA20 DA12 EA01 FA11 FA25

Claims (10)

[Claims] 1. An immunosuppressive protein adsorbent having a specific surface area of 0.1 square meter or more per gram and comprising a hydrophilic amine residue bound to a water-insoluble carrier. 2. The immunosuppressive protein adsorbent according to claim 1, wherein the hydrophilic amine residue is a quaternary ammonium group having 3 to 18 carbon atoms per nitrogen atom. 3. The immunosuppressive protein adsorbent according to claim 1, wherein the immunosuppressive protein is transforming growth factor beta. 4. The immunosuppressive protein adsorbent according to claim 1, wherein the immunosuppressive protein is an immunosuppressive acidic protein. 5. The immunosuppressive protein adsorbent according to claim 1, wherein the water-insoluble carrier is a polysulfone polymer. 6. The immunosuppressive protein adsorbent according to claim 1, wherein the water-insoluble polymer is poly (aromatic vinyl compound). 7. The immunosuppressive protein adsorbent according to claim 1, wherein the water-insoluble polymer having a hydrophilic amine residue bonded thereto is molded into a film, a fiber, a granular material or an assembly thereof. 8. The adsorbent according to claim 1, wherein the surface of any one of a membrane, a fiber and a granular material is coated with a water-insoluble polymer having a hydrophilic amino group bonded thereto. 9. An extracorporeal circulation column packed with the adsorbent according to claim 1. 10. An extracorporeal circulation column for cancer treatment, which is packed with the adsorbent according to claim 1.