JP4957886B2 - Column with immune activation ability - Google Patents

Description

  The present invention relates to a column having immune activation ability. In particular, the present invention relates to an extracorporeal circulation treatment column that can be used for the treatment of infectious diseases and cancer.

  In industrialized countries, the proportion of people who die from cancer is increasing rapidly with increasing lifespan. An increase in infectious diseases due to aging is also a problem. These are thought to be caused by a decrease in immune capacity due to aging. Anticancer agents are mainly used for tumor treatment. However, since anticancer agents destroy not only tumor cells but also normal bone marrow cells, the immune ability of patients is reduced. Therefore, the advent of a method that enhances immunity without lowering immunity is desired. One possible method is to introduce white blood cells outside the body and contact them with materials that enhance immunity.

  Such a cell activating material for extracorporeal circulation has been conceived before, and a fiber (non-patent document 1) in which lipopolysaccharide derived from the cell wall of Gram-negative bacteria is immobilized or porkwood mitogen, which is a kind of lectin, is immobilized. The bead (Non-patent Document 2) has been reported. However, since these ligands, lipopolysaccharide and porkwood mitogen, are both highly toxic substances, if they are packed in a column tube (cylinder) and subjected to extracorporeal circulation, these should be avoided. When the ligand is released from the carrier into the blood and travels throughout the body, the patient is at risk of shock. In addition, immunosuppressive substances that interfere with the activation of immunocompetent cells increase in the blood of cancer patients as the cancer progresses, so that immune cell activation does not sufficiently proceed with the immunostimulatory material alone.

As another treatment method, there is activated lymphocyte therapy (Non-patent Document 3) in which peripheral blood lymphocytes of a patient are cultured and expanded outside the body and then returned to the patient. is not. The reason is that there are proteins and cells that suppress immunity in the patient's blood as described above. In addition, the method of culturing in vitro is different from the method of performing extracorporeal circulation treatment using the above-mentioned column, and there is a risk of causing infections due to contamination with pathogenic bacteria during the culturing. Take it.
Therefore, a column having high immunostimulatory ability and excellent safety is desired.
Tani T, et al. Therapeutic Apheresis 4,167-172, 2000. Numa K, et al. Cancer Immunol Immunother, 32, 125-130, 1990. Lancet 2000: 356, 802-807

  In order to solve the above problems, an object of the present invention is to provide a column that can efficiently activate immunity and can be used effectively for infectious diseases and cancer treatment.

  In order to solve the above-mentioned problems, the present inventors have conducted various studies as to whether or not a column with high immunostimulatory ability and high safety and economy can be obtained. As a result, as a result of transforming growth factor-β (hereinafter referred to as TGF-). Columns packed with a material that removes immunosuppressive substances such as β (abbreviated as β) and immunosuppressive acidic proteins and a material that is made by immobilizing lipoteichoic acid on a water-insoluble carrier activates cellular immunity of rat splenocytes. The present invention has been found.

  That is, the present invention is a column comprising a material obtained by immobilizing lipoteichoic acid on a water-insoluble carrier and an immunosuppressive substance removing material as fillers. The column is suitable for use as a systemic circulation column.

  The lipoteichoic acid can be synthesized, but those derived from Gram-positive bacteria are preferred. Examples of lipoteichoic acid that are particularly excellent in safety include those derived from hemolytic streptococci and Bacillus subtilis.

  The immunosuppressive substance removing material is preferably a material that adsorbs TGF-β or immunosuppressive acidic protein.

  According to the present invention, a column with high immune activation ability can be obtained. In particular, a column for extracorporeal circulation treatment that can be used effectively for infectious diseases and cancer treatment is obtained.

  The material obtained by immobilizing lipoteichoic acid in the present invention on a water-insoluble carrier (hereinafter abbreviated as lipoteichoic acid immobilization material) is Gram-positive represented by hemolytic streptococci, Bacillus subtilis, Staphylococcus aureus, etc. It means a compound in which lipoteichoic acid, which is known as a compound existing on the cell wall of a fungus, is immobilized on a water-insoluble carrier by a chemical bond. Lipotycoic acid can be obtained in pure form by extracting bacterial cells with an organic solvent such as butanol, then decomposing / removing contaminating proteins, and chromatographic purification as appropriate (J. Immunotherapy 1993: 13: 232- 242).

In general, the water-insoluble carrier for fixing lipoteichoic acid is preferably composed of a polymer. Since the lipoteichoic acid has an amino group or a hydroxyl group in the molecule, the polymer needs to be a polymer (reactive polymer) having a functional group capable of forming a covalent bond with these amino group or hydroxyl group. is there.
Specific examples of the functional group include halomethyl groups represented by chloromethyl group, bromomethyl group, iodomethyl group and the like, and active halogen-containing groups such as haloacetyl groups represented by chloroacetyl group, bromoacetyl group, iodoacetyl group and the like. An isothiocyanate group, an isocyanate group, an aldehyde group, a carboxyl group and the like and derivatives thereof are preferably used.
As the polymer, polystyrene, vinyl aromatic compound polymer represented by polyvinyl toluene, aromatic polysulfone polymer, aromatic polyetherimide, aromatic polyimide polymer, polyacrylate polymer, polymethacrylic acid Examples include ester polymers, polyvinyl alcohol polymers, polyvinyl chloride, and glass.
Specific examples of the reactive polymer constituting the preferred water-insoluble carrier include a part of the aromatic nucleus of the aromatic compound polymer represented by the following general formula (1):
_{- (CH 2) n -A- (} CH 2) m -Y (1)
(In the formula, n represents an integer of 0 to 20, m represents an integer of 0 to 20, and n and m may be the same or different. A represents an oxygen atom, a sulfur atom, a nitrogen atom, urea. Group, amide group, or methylene group, and Y is Cl, Br, I, -N = C = O, -N = C = S).

  The molecular weight of the polymer (and copolymer) used in the carrier of the present invention is not particularly limited as long as it can be molded, but is usually from 50,000 to 5,000,000, particularly from the good moldability, Those of 100,000 to 1,000,000 are preferably used.

  The appropriate amount of functional groups in the polymer, that is, the functional group density varies depending on the chemical structure and application of the main polymer, but if it is too small, its function will not be expressed, and if it is too large, it will be used when lipoteichoic acid is fixed. It is left behind and wasted. Therefore, for example, in the case of an aromatic compound polymer having a functional group represented by the general formula (1), the amount of the functional group is usually 0.001 to 4 per repeating unit (monomer). 0.01-1 piece is preferable.

If the immobilization density of lipoteichoic acid is too small, its function is not expressed, and if it is too large, it is not used and is wasted. Therefore, the amount of lipoteichoic acid is usually preferably 0.0000001 to 0.04, particularly 0.00001 to 0.005 per repeating unit. However, when used as a filler for a column for extracorporeal circulation treatment, lipoteichoic acid immobilized on the surface of the material can be brought into contact with blood cells, so it works effectively, but the lipoteichoic acid present inside has an opportunity to come into contact with blood cells. Because there is no, it does not work effectively. Therefore, the density of the lipoteichoic acid on the surface of the lipoteichoic acid immobilization material is important. From this point of view, it is preferable to produce a reactive polymer molded product in a lipoteichoic acid solution and immobilize it rather than molding a polymer to which lipoteichoic acid is immobilized. In addition, it is manufactured by a method in which an existing molded product such as a fiber is immersed in a solution of a reactive polymer soluble in a solvent, and the reactive polymer is applied and then placed in a solution of lipoteichoic acid. A method is also preferred.
On the other hand, when used as an extracorporeal circulation treatment column, the volume of blood that can be safely removed from the body is said to be 200 mL in the case of humans, so there is a limit to the size of the column, in other words, the amount of material that can be filled. Considering these, the immobilization density of lipoteichoic acid is preferably 0.01 mg / g to 10 mg / g, and more preferably 0.1 mg / g to 1 mg / g from the viewpoint of efficient use of lipoteichoic acid.
The amount of lipoteichoic acid immobilized on the carrier can be easily determined from the amount of lipoteichoic acid remaining in the immobilization reaction mother liquor at the time of immobilization, but it can also be obtained by analyzing the lipoteichoic acid-immobilized material. is there.
In the latter method, the lipoteichoic acid-immobilized material is immersed in an anti-lipotycoic acid antibody aqueous solution to allow antigen-antibody binding to proceed, and then the amount of free antibody remaining in the aqueous solution is measured. It is possible to easily quantify by obtaining the difference between the two. In addition, after hydrolyzing the lipoteichoic acid-immobilized material with acid, glycerin, phosphoric acid, glucose, amino sugar, amino acid, long chain fatty acid, etc. contained in the hydrolyzed solution are analyzed by high performance liquid chromatography. It is also possible to quantify.

The production of the lipoteichoic acid-immobilized material of the present invention can be achieved by adding a water-insoluble carrier to the corresponding solution of lipoteichoic acid as it is or by adding a condensing agent as necessary. Specifically, when the carrier has an active halogen group such as a halomethyl group or a haloacetyl group, the immobilization reaction can be advanced by adjusting the pH of the lipoteichoic acid aqueous solution to 7-12. Moreover, when a support | carrier has an isothiocyanate group and an isocyanate group, it can advance by adding a tertiary amine as a catalyst to a reaction liquid. In this case, lipoteichoic acid is immobilized by a urea bond, a thioureido bond, or a thiourea bond or a thioureido bond. When the carrier has a carboxyl group, it can be obtained by adding a peptide condensing agent such as dicyclohexylcarbodiimide or water-soluble carbodiimide to the reaction solution.
The reaction solvent is not particularly limited as long as it dissolves lipoteichoic acid, but water, carriers such as dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone An organic solvent that swells is preferably used.

The immunosuppressive substance removing material referred to in the present invention includes TGF-β, immunosuppressive acidic protein, interleukin-6 (hereinafter abbreviated as IL-6), prostaglandin E2 (hereinafter abbreviated as PGE2), soluble Fas, and the like. It means a material that efficiently adsorbs a substance that suppresses immunity to cancer cells. In particular, since TGF-β strongly suppresses cellular immunity such as killer cells and natural killer cells, a material excellent in TGF-β adsorbability (particularly, a material capable of adsorbing latent TGF-β) is preferably used. It is done.
Specific examples of the immunosuppressive substance removing material include a porous water-insoluble carrier, N, N-dimethylbutylammonium group, N, N-dimethylhexylammonium group, N, N-dimethyloctylammonium group, N, N, Examples thereof include those having a hydrophilic amino group such as an N-triethylammonium group and an N, N, N-trin-propylammonium group.
The production method includes N, N-dimethylbutylamine, N, N-dimethylhexylamine, N, N-dimethyloctylamine, triethylamine, tri-n-propylamine groups, etc. on a porous water-insoluble carrier containing active halogen groups. It can be easily obtained by reacting. The reaction is accomplished by heating at room temperature to 100 ° C. for 1 to 10 hours using an organic solvent that swells a carrier such as dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone. it can. At this time, when a small amount of potassium iodide is allowed to coexist, the reaction rate increases.
Examples of the active halogen group-containing porous water-insoluble carrier include aromatic compound polymers such as a vinyl aromatic compound polymer having an active halogen group, an aromatic polysulfone polymer, a polyetherimide, and an aromatic polyimide polymer. . Specific examples thereof include chloroacetamidomethylated fibers obtained by amidomethylating polystyrene fibers with N-hydroxymethyl-2-chloroacetamide.

  The appropriate amount of the hydrophilic amino group in the polymer of the immunosuppressive substance-removing material according to the present invention, that is, the functional group density varies depending on the chemical structure and application of the main polymer. It is not expressed, and when it is too much, it is not used and is wasted. Therefore, the amount of the hydrophilic amino group is usually preferably 0.001 to 4, more preferably 0.01 to 1, per repeating unit (monomer).

As the configuration of the column according to the present invention, since immune cells are more likely to be activated after the immunosuppressive substance is removed, the immunosuppressive substance removing material is usually placed upstream of the column and the downstream side of the column. It is used by filling with a lipoteichoic acid immobilization material. The amount of the immunosuppressive substance removing material and the lipoteichoic acid immobilizing material to be filled is determined according to the patient's condition and therapeutic purpose.
When used as a column for extracorporeal circulation therapy, it is necessary to design so as to minimize the burden on the patient. Since the human blood volume is approximately one-third of the body weight, the internal volume of the systemic circulation column is preferably 8 mL or less per kg body weight. When filling an immunosuppressive substance removing material and a lipoteichoic acid immobilization material into a column tube for systemic circulation treatment, it is necessary to secure a blood flow path in the column. It becomes as follows. Since the fibers are bulky, the appropriate filling amount when using a fibrous carrier for both materials is 0.1 to 0.4 g, more preferably 0.1 to 0.3 g, per 1 mL of internal volume. The filling ratio of the immunosuppressive substance removing material and the lipoteichoic acid-immobilizing material is preferably 1: 4 to 4: 1 by weight when both are fibrous. When there are many immunosuppressive substances in the blood, it is preferable to increase the ratio of the immunosuppressive substance removing material.

  The shape of the lipoteichoic acid immobilization material and the immunosuppressive substance removing material of the present invention is a fiber, a membrane, a film, a hollow fiber, a nonwoven fabric, a granular material, or a higher-order processed product thereof, and is appropriately selected according to the application. . These can be used for cancer treatment in the form of extracorporeal circulation columns.

Next, the present invention will be specifically described based on examples. The concentration analysis of lipoteichoic acid in this example, the preparation of cancer-bearing rats, and the extracorporeal circulation of the rats were as follows.
1. Analysis of lipoteichoic acid concentration Determined by the phenol-sulfuric acid method. That is, 2 mL of an aqueous solution containing lipoteichoic acid and 1 mL of 5% phenol water were added to a glass test tube having a diameter of 20 mm, and then 5 mL of concentrated sulfuric acid was rapidly added and shaken. After allowing this solution to stand for 30 minutes, absorbance at 485 nm was measured, and the concentration was determined from a calibration curve. When preparing a calibration curve, when triethylamine was used as a reaction catalyst, a calibration curve was prepared by adding triethylamine obtained from the absorbance of the solution before the treatment to the same concentration.
2. Preparation of tumor-bearing rats 4-Dimethylaminoazobenzene-induced hepatoma cell KDH-8 {Akira Yano, Hokkaido Medical Journal, Vol. 68, No. 65, 664-664 (1993)} was suspended in phosphate buffered saline 2 × 10 6 A tumor-bearing rat was prepared by inoculating 0.5 mL of a solution at a concentration of 5 cells / mL subcutaneously on the back of a WKAH / Hkm rat (male, 10 weeks old).
3. Rat extracorporeal circulation (column for extracorporeal circulation treatment)
A column for extracorporeal circulation treatment was prepared by filling an immunosuppressive substance-removing material on the column inlet side and a lipotycoic acid immobilization material on the outlet side. After 70% alcohol was passed through the column and circuit for sterilization, 15 mL of heparinized physiological saline (15 units / mL) was allowed to flow at a rate of 2 mL / min immediately before extracorporeal circulation.
(Extracorporeal circulation)
A tumor-bearing rat weighing about 350 g is anesthetized with Nembutal, cannulated into the artery and vein of the left femur, exsanguinated from the artery, passed through the extracorporeal circulation column using a microtube pump, and returned to the vein. Blooded. Extracorporeal circulation was performed for 1 h at a blood flow rate of 2 mL / min. Heparin in the extracorporeal circulation was continuously administered at 100 units / hour.

[Example 1]
(Production of column)
1. Preparation of water-insoluble carrier raw yarn 36 island-island composite fibers, each of which is made of a core-sheath composite, were obtained under the spinning conditions of spinning speed of 800 m / min and draw ratio of 3 times using the following components. .
Island core component; Polypropylene island sheath component: Polystyrene 90% by weight, polypropylene 10% by weight
Sea component: ethylene terephthalate unit as a main repeating unit, and copolymerized polyester composite ratio (weight ratio) containing 3 wt% of 5-sodiumsulfoisophthalic acid as a copolymer component; core: sheath: sea = 40: 40: 20
The sea component of this fiber 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 polypropylene reinforced polystyrene fiber.

2. Preparation of water-insoluble carrier 1 To a mixed solution of 700 mL of nitrobenzene and 460 mL of sulfuric acid, 3.6 g (0.2%) of paraformaldehyde was added, dissolved at 20 ° C., cooled to 0 ° C., and 127 g (7%) of N- Methylol-α-chloroacetamide was added and dissolved at 5 ° C. or lower. 39 g of the above-mentioned raw yarn 1 was immersed in this, and left still for 2 hours at room temperature. Thereafter, the fiber was taken out, placed in a large excess of cold methanol and washed. The fiber was thoroughly washed with methanol, washed with water, and dried to obtain 57.5 g of α-chloroacetamidomethylated polystyrene fiber (water-insoluble carrier 1) (functional group density per repeating unit; 1).

3. Preparation of water-insoluble carrier 2 13 g of water-insoluble carrier 1 was immersed in a solution of 20 g of sodium thiocyanate in 400 mL of dimethylformamide and immersed at room temperature for 48 hours. The fiber was taken out, washed with water, and vacuum-dried to obtain 13 g of α-thioisocyanatoacetamidomethylated polystyrene fiber (water-insoluble carrier 2).

4). Preparation of lipoteichoic acid immobilization material 1
10 mg of lipoteichoic acid derived from Streptococcus pyrogenes (Sigma) was dissolved in PBS to make 100 mL, 6.0 g of water-insoluble carrier 2 was added thereto, shaken for 1 h at room temperature, 0.2 mL of triethylamine was added, and 48 h at room temperature. Shake. The fiber was taken out and washed with water to obtain a lipoteichoic acid immobilization material 1. The amount of immobilization was calculated from the difference in the lipoteichoic acid concentration of the aqueous solution before and after immobilization. The immobilization density was 0.61 mg / g.

5. Preparation of immunosuppressive substance removing material 1 5 g of water-insoluble carrier 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 bath at 80 ° C. for 3 hours. The water-insoluble carrier was taken out, immersed in a 1 mol / L saline solution, washed with water, and dried under vacuum to obtain 7.8 g of dimethylhexylammonium-containing fiber (immunosuppressive substance removing material 1) (repeatedly). Functional group density per unit; 0.92).

6). Preparation of column 1 200 mg of immunosuppressive substance-removing material 1 is packed on the column entrance side and 60 mg of Streptococcus pyrogenes-derived lipoteichoic acid-immobilized material 1 is packed on the exit side of a polypropylene cylindrical column tube having an inner diameter of 1 cm and an internal volume of 2 ml. A column for extracorporeal circulation treatment (column 1) was prepared.

[Example 2]
(Examination of effects of extracorporeal circulation treatment)
Seven days after tumor inoculation, four cancer-bearing rats weighing about 350 g were treated with extracorporeal circulation using column 1. Extracorporeal circulation was performed for 1 h at a blood flow rate of 2 mL / min. During the extracorporeal circulation, heparin 100 units / h was continuously administered.
One out of four tumors disappeared and was completely cured. The average survival time of the remaining 3 animals was 64.0 ± 1.9 days.
In contrast, the average survival time of 4 untreated cancer-bearing rats was 54.8 ± 3.0 days.

[Comparative Example 1]
Extracorporeal circulation treatment was performed using a column filled only with lipoteichoic acid immobilization material, and the effect was examined.
1. Preparation of Comparative Column 1 An extracorporeal circulation treatment column (Comparative Column 1) was prepared by filling 60 mg of the above lipoteichoic acid immobilization material 1 into a polypropylene cylindrical column tube having an inner diameter of 1 cm and an internal volume of 2 ml.
2. Preparation of tumor-bearing rats and treatment of extracorporeal circulation Seven days after tumor inoculation, three cancer-bearing rats weighing about 350 g were treated with extracorporeal circulation using comparative column 1. Extracorporeal circulation was performed for 1 h at a blood flow rate of 2 mL / min. During the extracorporeal circulation, heparin 100 units / h was continuously administered.
The average survival time of 4 animals was 56.7 days.

[Comparative Example 2]
An extracorporeal circulation treatment was performed using a column filled with only an immunosuppressive substance removing material, and the effect was examined.
1. Preparation of Comparative Column 2 An extracorporeal circulation treatment column (Comparative Column 2) was prepared by filling 0.2 g of an immunosuppressive substance removing material 1 into a polypropylene cylindrical column tube having an internal diameter of 1 cm and an internal volume of 2 ml.
2. Preparation of tumor-bearing rats and treatment of extracorporeal circulation Seven days after tumor inoculation, three cancer-bearing rats weighing about 350 g were treated with extracorporeal circulation using comparative column 2. Extracorporeal circulation was performed for 1 h at a blood flow rate of 2 mL / min. During the extracorporeal circulation, heparin 100 units / h was continuously administered.
The average survival time of 4 animals was 55.3 days.

From the above results, it was confirmed that the column according to the present invention was effective for extracorporeal circulation treatment.

Claims (7)

A column having a material obtained by immobilizing lipoteichoic acid on a water-insoluble carrier and an immunosuppressive substance removing material as fillers, the immunosuppressive substance removing material being packed upstream of the column, and the lipoteicho being downstream. A column filled with a material formed by immobilizing an acid .
  The column according to claim 1, wherein the lipoteichoic acid is derived from a Gram-positive bacterium.   The column according to claim 1, wherein the lipoteichoic acid is derived from hemolytic streptococci.   The column according to claim 1, wherein the lipoteichoic acid is derived from Bacillus subtilis.   The column according to any one of claims 1 to 4, wherein the immunosuppressive substance removing material is a material that adsorbs transforming growth factor-β.   The column according to any one of claims 1 to 5, wherein the immunosuppressive substance removing material is a material that adsorbs an immunosuppressive acidic protein.   The column according to any one of claims 1 to 6, which is used for extracorporeal circulation therapy.