JP2008007901A - Immunocyte activating material and column - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material activating an immunocyte and having excellent safety for living bodies, to provide a column filled with the material and to provide a method for effectively using the column. <P>SOLUTION: The immunocyte activating material is characterized by immobilizing lipoteichoic acid on a water-insoluble carrier having a functional group covalently bonding to an amino group or a hydroxy group. The column is filled with the immunocyte activating material. The method for using the column is characterized by using the column for extracorporeal circulation in combination with administration of an anti-malignant tumor agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an immune cell activator and a column that can be used for treating infectious diseases and cancer, and a method of using the column.

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. Anti-neoplastic agents are mainly used for the treatment of tumors, but anti-neoplastic agents destroy not only tumor cells but also normal bone marrow cells, leading to a decrease in the immune capacity of patients.
For example, neutropenia often occurs as a side effect of administration of an antineoplastic agent, and in this case, the risk of opportunistic infection increases. For this prevention, granulosite colony stimulating factor (G-CSF) is administered, but it is not effective for metastasis and growth of cancer, and conversely, there is a risk of promoting cancer growth. is there. In addition, not only neutrophils but also other immune cells such as lymphocytes are damaged by administration of the antineoplastic agent, so that not only the pathogenic bacteria but also the protective ability against cancer is reduced.

  Therefore, a method for enhancing the immunity of a patient whose immunity has decreased due to aging or administration of an antineoplastic agent is desired. In particular, in cancer patients undergoing chemotherapy, the emergence of methods that reduce the amount of antineoplastic agent used to prevent damage to the immune system and activate immune cells to enhance immunity is desired.

As a method for activating immunity, a method is conceivable in which leukocytes are guided outside the body and contacted with a material that enhances 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, these ligands, lipopolysaccharide and porkwood mitogen, are both highly toxic substances. If these are packed in the column body (cylinder) and used for extracorporeal circulation, they should be removed from the carrier. If released to the whole body, there is a risk of shock to the patient.
Therefore, there is a need for an immunostimulatory material with better safety.
Tani T, et al. Therapeutic Apheresis 4,167-172, 2000. Numa K, et al. Cancer Immunol Immunother, 32, 125-130, 1990.

  In order to solve the above problems, an object of the present invention is to provide a material that can activate immune cells and is excellent in safety to living bodies, and a column packed with the material. Another object of the present invention is to provide a method for using the column that can reduce the dose of an antineoplastic agent.

  In order to solve the above-mentioned problems, the present inventors have made various studies as to whether an immunostimulatory material that can be used safely is obtained. As a result, low-toxic lipoteichoic acid is converted to its amino group or hydroxyl group. The inventors have found that a material immobilized on a water-insoluble carrier by covalent bonding activates cellular immunity of rat splenocytes, and have reached the present invention.

  That is, the present invention is an immune cell activating material obtained by immobilizing lipoteichoic acid on a water-insoluble carrier having a functional group capable of being covalently bonded to an amino group or a hydroxyl group.

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

  The functional group that can be covalently bonded to the amino group or the hydroxyl group is preferably at least one selected from the group consisting of an active halogen group, an isothiocyanate group, an isocyanate group, an aldehyde group, and a carboxyl group.

  The water-insoluble carrier is preferably an aromatic compound polymer.

  Moreover, it is preferable that the shape of the said immune cell activation material is a fiber, a film | membrane, a hollow fiber, or a granular form. The activator is suitable for use in a packed column. The column can also be used as a systemic circulation column.

  Furthermore, as a result of studying more effective methods of using the extracorporeal circulation column, the present inventors have used the anti-neoplastic agent together with an anti-neoplastic agent in the case of cancer treatment, thereby increasing the dosage of the anti-neoplastic agent. The present invention has been completed.

  That is, the present invention is a method for using the extracorporeal circulation column, which is used for extracorporeal circulation in combination with administration of an antineoplastic agent (that is, before or after administration of an antineoplastic agent or simultaneously with administration). It is the usage method of the column characterized by doing. A more preferred method of use is a method of use for extracorporeal circulation after administration of an antineoplastic agent.

  The antineoplastic agent is preferably an antimetabolite antineoplastic agent, more preferably gemcitabine.

  According to the immune cell activator of the present invention, cell activation associated with induction of killer cells can be caused. Further, according to the method of use of the present invention, it is possible to treat advanced cancer or to prolong the life and improve the quality of life of patients.

  The functional group that can be covalently bonded to an amino group or a hydroxyl group in the present invention is not particularly limited as long as it is a functional group that can chemically react with an amino group or a hydroxyl group to form a covalent bond. Specific examples include halomethyl groups represented by chloromethyl group, bromomethyl group, iodomethyl group, etc., active halogen groups represented by haloacetyl groups represented by chloroacetyl group, bromoacetyl group, iodoacetyl group, etc. Examples include an isocyanate group, an isocyanate group, an aldehyde group, a carboxyl group, and derivatives thereof, and one or more selected from these groups are appropriately used depending on the purpose.

The water-insoluble carrier referred to in the present invention is a polystyrene, a vinyl aromatic compound polymer represented by polyvinyltoluene, an aromatic polysulfone polymer, a polyetherimide, an aromatic polyimide polymer, a polyacrylate polymer, Molding of polymers such as methacrylic acid ester polymers, polyvinyl alcohol polymers, polyvinyl chloride, glass, etc., which are substantially water-insoluble polymers and can fix lipoteichoic acid with chemical bonds. Means goods.
As a specific example, in the case of an aromatic compound polymer, a part of the aromatic nucleus is represented by the following general formula (1):
_{- (CH 2) n -A- (} CH 2) m -Y (1)
(In the formula, n represents an integer of 1 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 of the present invention, that is, the 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 not be used. become. 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.

  Lipotycoic acid is a compound that is present in the cell walls of Gram-positive bacteria such as hemolytic streptococci, Bacillus subtilis, and Staphylococcus aureus. And, if appropriate, chromatographic purification can be obtained in pure form (eg J. Immunotherapy 1993: 13: 232-242).

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 an extracorporeal circulation material, lipoteichoic acid immobilized on the surface of the immune cell activator can work with blood cells, so it works effectively, but lipoteichoic acid present inside the immune cell activator is blood cells. Because there is no opportunity to contact with, it does not work effectively. Therefore, the density on the surface of the molded product is important for the immobilization density of lipoteichoic acid. From this point of view, it is preferable to use a method in which a water-insoluble carrier having a functional group is formed and then placed in a lipoteichoic acid solution and immobilized.
On the other hand, the amount of blood that can be safely taken out of the body during extracorporeal circulation is said to be 200 mL for humans, so there is a limit to the size of the column, and therefore the amount of activation material that can be packed is limited. . Taking these into consideration, the immobilization density of lipoteichoic acid in the immune cell activator produced by the above method is preferably 0.01 mg / g to 10 mg / g, and 0.1 mg / g from the viewpoint of efficient use of lipoteichoic acid. More preferred is ˜1 mg / g.
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 the lipoteichoic acid-immobilized product (immunocyte activation material) is analyzed. It is also possible to ask.
As the latter method, the lipoteichoic acid-immobilized product 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 can be easily quantified by obtaining the difference. In addition, after hydrolyzing lipoteichoic acid immobilized product 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 etc. It is also possible to do.

The immune cell activator of the present invention can be produced by adding a carrier to the corresponding solution of lipoteichoic acid and reacting it as it is or by adding a condensing agent as necessary. Specifically, the carrier has an active halogen group typified by a halomethyl group typified by a chloromethyl group, a bromomethyl group, an iodomethyl group, etc., a haloacetyl group typified by a chloroacetyl group, a bromoacetyl group, an iodoacetyl group, etc. If so, 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 a carrier such as dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone can be used. An organic solvent for swelling is preferably used.

  The immune cell activation referred to in the present invention is the activation of cellular immunity, which means the proliferation and activation of killer cells having the role of extinguishing cancer cells and virus-infected cells. Second is the activation of humoral immunity, which means the activation of cells involved in the production of antibodies against cancer cells and viruses. Cellular immunity is said to be particularly important in the treatment of cancer. It is known that interleukin-12 (hereinafter referred to as IL-12) and interferon-γ have an important role in the growth and activation of this killer cell, so that IL-12 and interferon-γ can be induced. Is an important function of immune cell activator. The mechanism of action of the present invention is not clear, and further investigation is necessary to clarify this, but it is considered that dendritic cells and their toll-like receptors are involved in the early stage of activation. It is done.

  The shape of the immunologically active 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 depending on the application. These can be packed in an extracorporeal circulation column and used for cancer treatment. In this case, it is effective to use in combination with an antineoplastic agent.

  The antineoplastic agent used in the present invention is not particularly limited as long as it is a drug that exhibits antitumor properties when used alone. Specific examples include antimetabolite antitumor agents represented by gemcitabine, fluorouracil, tegafur, cytarabine, methotrexate, alkylating agents represented by cyclophosphamide, vincristine, vinblastine, vindesine, etoposide, irinotecan, Alkaloid antineoplastic agents typified by docetaxel, paclitaxel, etc., antibiotic antineoplastic agents typified by doxorubicin, epirubicin, biralubicin, daunorubicin, mitomycin C, actinomycin D, peplomycin, neocalcinostatin, bleomycin, etc. Enzyme-inhibiting antineoplastic agents such as gefitinib, cisplatin and carboplatin. Of these, antimetabolite anti-neoplastic agents are preferred because they do not have the carcinogenicity observed in alkylating agents and have low toxicity to bone marrow. Furthermore, among antimetabolite anti-neoplastic agents, gemcitabine is particularly preferable because it has a slow metabolism in tumor cells and thus has an antitumor effect and exhibits an antitumor effect on many solid tumors.

  The preferred administration method of the antineoplastic agent in the present invention includes a method of injecting into a tissue near the tumor, a method of injecting intravenously, a method of injecting intramuscularly, a method of administering orally, etc., and is not particularly limited. However, it is preferable to employ appropriately depending on the characteristics of the drug. If the dose is too small, the effect of the present invention is low, and if the dose is too large, bone marrow suppression or the like occurs, the induction of tumor immunity is suppressed, and the antitumor effect is reduced. In general, it is desirable to use 1/100 to 1/2 of the appropriate dose designated for each antineoplastic agent. The administration timing of the antineoplastic agent may be before or after the extracorporeal circulation treatment, but is more preferably performed prior to the extracorporeal circulation treatment. Particularly preferably, it is performed 24 hours to 200 hours before the extracorporeal circulation treatment, more preferably 24 hours to 100 hours.

  The surface area of the immune cell activator used in the present invention is preferably 0.1 square meter or more, more preferably 1 square meter or more per gram of the molded product. 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).

The extracorporeal circulation column used in the present invention is prepared by, for example, using an immune cell activator in which a lipoteichoic acid in the form of cotton, tube braid, or felt is immobilized so that the void volume is about 200 mL or less. This can be achieved by filling the cylindrical column body.
As a basic implementation method of extracorporeal circulation, a puncture catheter for blood collection, a drip chamber connected with an infusion pump for continuously administering an anticoagulant, a blood pump, a drip chamber, the extracorporeal circulation column of the present invention, and a drip chamber The blood return puncture catheter can be connected in the order of a tube having an appropriate thickness to produce an extracorporeal circuit, and blood can be allowed to flow therethrough. Blood is collected and returned by puncturing the artery or vein of the thigh or arm. For large mammals, extracorporeal circulation devices and blood circuits that are commercially available for hemodialyzers and adsorption blood purifiers can be used. The extracorporeal circulation is preferably performed for 10 to 300 minutes, usually 30 to 120 minutes. The anticoagulant used for extracorporeal circulation is not particularly limited, and heparin, fusan, etc. are used.

  In the treatment according to the method of use of the present invention, since the amount of the antineoplastic agent (anticancer agent) used is small, there are advantages in that there are few side effects. Further, there is an advantage that the patient's QOL is not lowered.

The present invention will be specifically described based on examples. The analysis of the lipoteichoic acid concentration and NK activity in this example was 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. Measurement of NK activity (Preparation of YAC-1 cells)
YAC-1 cells, which are cancer cells sensitive to NK cells, contain penicillin G at 50 units / mL, streptomycin at 50 microgram / mL, 2-mercaptoethanol at 50 micromol / L, and fetal bovine serum at 10% by volume. Subculture was performed in a PRMI1640 medium (hereinafter abbreviated as a complete medium) added to the above.
(Rat splenocytes)
After anesthetizing the rat with Nembutal, blood was removed from the abdominal aorta and sacrificed, and the spleen was collected. The spleen was finely broken in a complete medium, and the cells were collected, and then treated with a low osmotic pressure solution to remove red blood cells and hemolyzed. The obtained cells were suspended in a complete medium to obtain a spleen cell solution.
(Measurement of europium label and NK activity of YAC-1 cells)
Europium labeling of YAC-1 cells was performed according to the literature (Nagao F, Tanaka M, 1992 Medical Immunology, 23 (1), 84-88). Spleen cells and europium-labeled YAC-1 cells were mixed at 40: 1, 20: 1, 10: 1, 5: 1 (cell number ratio) and then cultured in a carbon dioxide incubator at 37 ° C. for 20 hours. . Europium elution was measured using a Wallac ARVO ^™ SX DELFIA 1420 multilabel counter.

[Example 1]
(Preparation of water-insoluble carrier 1)
1. Preparation of raw yarn 36 islands of sea-island composite fiber, in which the island is further formed of a core-sheath composite, was obtained using the following ingredients under the spinning conditions of spinning speed of 800 m / min and draw ratio of 3 times.
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 ternary composite fiber obtained above was heated in an aqueous caustic soda solution to dissolve the sea component, and a core yarn 1 having a diameter of 4 μm was obtained as a core-sheath polypropylene reinforced polystyrene fiber.
2. Preparation of water-insoluble carrier To a mixed solution of 700 mL of nitrobenzene and 460 mL of sulfuric acid, 3.6 g (0.2 wt%) of paraformaldehyde was added, dissolved at 20 ° C., cooled to 0 ° C., and 127 g (7 wt%) 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).

[Example 2]
(Preparation of water-insoluble carrier 2)
13 g of water-insoluble carrier 1 was immersed in a solution of 20 g of sodium thiocyanide 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).

[Example 3]
(Preparation of water-insoluble carrier 3)
After cooling a mixed solution of 16 mL of nitrobenzene and 32 mL of sulfuric acid to 0 ° C., 4.2 g of N-hydroxymethyl-2-chloroacetamide was added and dissolved, and this was dissolved in 1.5 L of nitrobenzene of Udelpolysulfone P3500 at 10 ° C. The solution (150 g / 1.5 L) was added with good stirring, and further stirred at room temperature for 3 hours. The reaction mixture was then placed in a large excess of cold methanol to precipitate the polymer. The precipitate was washed well with methanol and dried to obtain 150 g of 2-chloroacetamidomethylated polysulfone (functional group density per repeating unit: 0.05; polymer-A).
10 g of Polymer-A was dissolved in 500 mL of tetrahydrofuran to obtain a polymer-A solution. In this solution, 40.0 g of nylon-6 cylinder braid having a single yarn fineness of 0.7 denier was immersed. After 20 hours, the tubular knitting was taken out, drained, air dried, and further vacuum dried to obtain 40.5 g of a coated knitted fabric (water-insoluble carrier 3).

[Example 4]
(Preparation of water-insoluble carrier 4)
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-hydroxy-2-chloroacetamide was added and dissolved at 5 ° C. or lower. 10 g of the original yarn 1 prepared in Example 1 was dipped 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, then washed with water and dried to obtain 15.0 g of polypropylene-reinforced 2-chloroacetamidomethylated polystyrene fiber (water-insoluble carrier 4).

[Example 5]
(Preparation of immune cell activation material 1)
Fixation of lipoteichoic acid 1
10 mg of lipoteichoic acid (Sigma) derived from Bacillus subtilis was dissolved in a carbonate buffer (pH 8.3) to make 100 mL, and 5.45 g of water-insoluble carrier 1 was added thereto, followed by shaking at room temperature for 48 hours. The fiber was taken out and washed with water to obtain an immune cell activation material 1 according to the present invention. The amount of immobilization was calculated from the difference in the lipoteichoic acid concentration of the aqueous solution before and after immobilization. The lipoteichoic acid immobilization density was 0.56 mg / g (92 pmol / g).

[Example 6]
(Preparation of immune cell activation material 2)
Fixation of lipoteichoic acid 2
10 mg of lipoteichoic acid (Sigma) derived from Bacillus subtilis was dissolved in a carbonate buffer (pH 9.1) to make 100 mL, and 5.55 g of water-insoluble carrier 1 was added thereto and shaken at room temperature for 48 h. The fiber was taken out and washed with water to obtain an immune cell activating material 2 according to the present invention. The amount of immobilization was calculated from the difference in the lipoteichoic acid concentration of the aqueous solution before and after immobilization. The lipoteichoic acid immobilization density was 0.61 mg / g (100 pmol / g).

[Example 7]
(Preparation of immune cell activation material 3)
Fixation of lipoteichoic acid 3
10 mg of lipoteichoic acid derived from Bacillus subtilis (Sigma) was dissolved in a carbonate buffer (pH 10.0) to make 100 mL, and 5.25 g of water-insoluble carrier 1 was added thereto and shaken at room temperature for 48 h. The fiber was taken out and washed with water to obtain an immune cell activation material 3 according to the present invention. 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 of lipoteichoic acid was 0.63 mg / g (103 pmol / g).

[Example 8]
(Preparation of immune cell activation material 4)
Fixation of lipoteichoic acid 4
10 mg of lipoteichoic acid (Sigma) derived from Streptococcus pyrogenes was dissolved in phosphate buffered saline (pH 7.4) to 100 mL, 6.0 g of water-insoluble carrier 1 was added thereto, and the mixture was shaken at room temperature for 48 hours. The fiber was taken out and washed with water to obtain an immune cell activation material 4 according to the present invention. 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 0.22 mg / g (37 pmol / g).

[Example 9]
(Preparation of immune cell activation material 5)
Fixation of lipoteichoic acid 5
10 mg of lipoteichoic acid derived from Streptococcus pyrogenes (Sigma) was dissolved in PBS to 100 mL, 6.0 g of water-insoluble carrier 2 was added thereto, shaken at room temperature for 1 h, added with 0.2 mL of triethylamine, and then at room temperature for 48 h. Shake. The fiber was taken out and washed with water to obtain an immune cell activation material 5 according to the present invention. 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 (100 pmol / g).

[Example 10]
(Preparation of immune cell activation material 6)
Fixation of lipoteichoic acid 6
10 mg of lipoteichoic acid (Sigma) derived from Bacillus subtilis was dissolved in carbonate buffer (pH 10.0) to make 100 mL, and 5.25 g of water-insoluble carrier 3 was added thereto, and shaken at room temperature for 48 h. The knitted fabric was taken out and washed with water to obtain an immune cell activation material 6 according to the present invention. The amount of immobilization was calculated from the difference in the lipoteichoic acid concentration of the aqueous solution before and after immobilization. The lipoteichoic acid immobilization density was 0.25 mg / g (41 pmol / g).

[Example 11]
(Preparation of immune cell activation material 7)
Fixation of lipoteichoic acid 7
10 mg of lipoteichoic acid (Sigma) derived from Streptococcus pyrogenes was dissolved in carbonate buffer (pH 9.1) to make 100 mL, and 5.55 g of water-insoluble carrier 4 was added thereto and shaken at room temperature for 48 h. The fiber was taken out and washed with water to obtain an immune cell activating material 7 according to the present invention. The amount of immobilization was calculated from the difference in the lipoteichoic acid concentration of the aqueous solution before and after immobilization. The lipoteichoic acid immobilization density was 0.56 mg / g.

[Example 12]
(Preparation of column 1)
An extracorporeal circulation column 1 according to the present invention was prepared by filling 0.15 g of the immune cell activator 5 into a polypropylene cylindrical column body having an inner diameter of 1 cm and an internal volume of 2 ml. Column 1 was pre-washed with a physiological saline containing 1000 units of heparin sodium immediately before extracorporeal circulation, and further washed with 500 mL of physiological saline.

[Example 13]
(Preparation of column 2)
An extracorporeal circulation column 2 according to the present invention was prepared by filling a cylindrical cylindrical column body made of polypropylene having an inner diameter of 1 cm and an internal volume of 2 ml with 0.15 g of the immune cell activator 7. Prior to extracorporeal circulation, pre-washed with 15 mL of physiological saline containing sodium heparin at a concentration of 10 units / mL.

[Example 14]
(Examination of effect of immune cell activator)
Using the immune cell activator 5, the effect of the immune cell activator according to the present invention was examined by an in vivo test using rat splenocytes.

Examination of the activity effect of rat spleen cells 4-dimethylaminoazobenzene-induced hepatocarcinoma cells KDH-8 {Yano Satoshi, Hokkaido Medical Journal, Vol. 68, No. 5, 654-664] subcutaneously on the back of WKAH / Hkm rats (male, 10 weeks old) (1993)} was inoculated 1 × 10 ⁶ to prepare tumor-bearing rats. Two weeks after tumor inoculation (tumor volume: 3.2 mL), the spleen was collected, and complete medium (RPMI1400 medium: containing 10% by volume of fetal calf serum, containing 2-mercaptoethanol 50 microgram / L, streptomycin 50 microgram / mL). Containing, penicillin-G50 units / mL), and after removing blood cells, erythrocytes were hemolyzed and a spleen cell solution (2 × 10 ⁶ cells / mL concentration) suspended in a complete medium was prepared. . Spleen cell fluid (2 × 10 ⁶ cells / mL concentration) was prepared in the same manner from normal rats of the same week that were not inoculated with cancer cells.

After 0.1 g of the immune cell activation material 5 according to the present invention was shaken in 10 mL of a complete medium at 37 ° C. for 1 h, the immune cell activation material was separated from the complete medium (hereinafter referred to as a washing medium), The mixture was shaken for 1 h at 37 ° C. in 5 mL of two types of spleen cell solutions. After removing the immune cell activator, the spleen cell solution was cultured for 48 hours in a 37 ° C. carbon dioxide incubator.
For comparison, a spleen cell alone cultured in a complete medium and a spleen cell suspended in a washing medium were prepared. The culture was started at a spleen cell concentration of 2 × 10 ⁶ cells / mL. Six types of cell cultures were centrifuged to collect the culture supernatant, and the cytokine concentration was measured.
Supernatant 1: Culture supernatant of tumor-bearing rat spleen cells after treatment with activating material 1h in supernatant of spleen cell supernatant 2: Culture supernatant of tumor-bearing rat spleen cells in complete medium Culture supernatant in washing medium Supernatant 4: Culture supernatant in splenocyte of normal rat splenocytes after treatment with activating material 1h Supernatant 5: Culture supernatant in normal medium of normal rat splenocytes Supernatant 6: Normal rat Culture supernatant in washing medium of splenocytes

表１から明らかなように、本発明に係る免疫細胞活性化材がキラー細胞の誘導に重要な役割を持つＩＬ−１２を強力に産生させることが分かる。 The measurement results of IL-12 are shown in Table 1.

As is apparent from Table 1, it can be seen that the immune cell activator according to the present invention strongly produces IL-12 having an important role in the induction of killer cells.

[Example 15]
(Examination of extracorporeal circulation treatment effect)
By in vivo tests using rats, extracorporeal circulation treatment was performed using column 1 according to the present invention, and the effect of extending survival time was examined.

Preparation of tumor-bearing rats and treatment of extracorporeal circulation Cancer-bearing model rats were prepared by inoculating 1 × 10 ⁶ KDH-8 cells subcutaneously in the back of 11 12-week-old WKAH: Hkm rats (male). For 4 rats 7 days after KDH cell inoculation, 24G × 3/4 ”Surfflow indwelling needle (Terumo Corp.) was inserted and fixed in the right femoral artery and right femoral vein, and microtube pump N-100 (Tokyo Science) Instrument Co., Ltd.) and column 1 were connected in series to create a circuit, which was circulated extracorporeally for 60 minutes at a blood flow rate of 2 ml / min.Heparin sodium injection solution (Takeda Pharmaceutical Co., Ltd.) ) Was continuously infused at a rate of 100 U / h, and the mean survival days after tumor inoculation in the extracorporeal circulation treatment group were 64.5 ± 1.5 days, whereas tumors of cancer-bearing rats that had not been treated with extracorporeal circulation The average survival time after inoculation was 55.7 ± 2.7 days, and the significant increase in survival days was observed (t test; p <0.001). Ensure that the column is effective in extending survival. I was able to confirm.

[Example 16]
(Examination of extracorporeal circulation treatment effect)
By an in vivo test using rats, extracorporeal circulation treatment was performed using column 1 according to the present invention, and the effect on NK activity was examined.
(Preparation of cancer-bearing rats and treatment of extracorporeal circulation)
Cancer-bearing model rats were prepared by inoculating 1 × 10 ⁶ KDH-8 cells subcutaneously in the back of four 10-week-old WKAH: Hkm rats (male). About 2 rats 11 days after KDH cell inoculation, 24G × 3/4 ”Surfflow indwelling needle (Terumo Corp.) was inserted and fixed in the right femoral artery and right femoral vein, and microtube pump N-100 (Tokyo Science) Instrument Co., Ltd.) and column 1 were connected in series to create a circuit, and extracorporeal circulation was performed for 60 minutes at a blood flow rate of 2 ml / min to form an extracorporeal circulation treatment group.Heparin sodium injection during extracorporeal circulation The solution (Takeda Pharmaceutical Co., Ltd.) was continuously injected at a rate of 100 U / h 25 days after tumor inoculation, spleen cells were removed from 2 rats in the extracorporeal circulation treatment group and 2 rats in the non-treatment group to obtain YAC-1 cells. The average of the NK activity of two animals in each group is shown in Fig. 1. The figure shows that the NK activity in the extracorporeal circulation treatment group is high.

[Example 17]
(Examination of effects when used in combination with antineoplastic agents)
The effect of combined treatment of column 2 and an antineoplastic agent according to the present invention was examined by an in vivo test using rats.

1. Preparation of tumor-bearing rats After anesthetizing a 12-week-old WKAH: Hkm rat (male) with an inhalation anesthetic sevofurene (manufactured by Maruishi Pharmaceutical Co., Ltd.), 4-dimethylaminoazobenzene-induced hepatoma cell KDH-8 {Yano Satoshi, Hokkaido Medical Journal, Vol. 68 No. 5, 654-664 (1993)} was 1 × 10 ⁶ cells inoculation. Tumors engrafted with 100% probability.

2. 7 days after cancer cell inoculation (rat weight: 400 to 430 g), 0.6 mg of gemcitabine hydrochloride (Japan Eli Lilly Co., Ltd., injection product dissolved in physiological saline and used as 20 mg / mL) Injection near the tumor.

3. Extracorporeal Circulation Treatment Of 12 cancer-bearing rats, 4 were treated without treatment and 4 were treated with only antineoplastic agents. The remaining 4 animals were subjected to extracorporeal circulation treatment 9 days after KDH cell inoculation (2 days after administration of the antineoplastic agent) and set as the extracorporeal circulation treatment group after administration of the antineoplastic agent. A 24G x 3/4 "Surfflow indwelling needle (Terumo Corp.) is inserted and fixed in the right femoral artery and right femoral vein, and the microtube pump N-100 (Tokyo Science Instrument Co., Ltd.) and the above column 2 are connected in series. The blood was collected from the femoral artery, returned to the femoral vein, and returned to the femoral vein for 1 hour at a blood flow rate of 2 ml / min.Heparin sodium during extracorporeal circulation An injection solution (Takeda Pharmaceutical Co., Ltd.) was continuously injected at a rate of 100 units / h.

The average survival time after tumor inoculation in 4 untreated groups was 50.3 ± 1.7 days.
The mean survival days after tumor inoculation of the 4 treatment groups administered with the antineoplastic agent were 61.5 ± 3.3 days.
The mean survival time after tumor inoculation of the 4 extracorporeal circulation treatment groups after administration of the antineoplastic agent was 71.5 ± 6.2 days.

  The statistically significant difference in mean survival days (t test; p <0.001) was P <0.01 between the no-treatment group and the extracorporeal circulation treatment group after administration of the antineoplastic agent, and the anti-neoplastic agent administration treatment group And P <0.05 between the group treated with the antineoplastic agent after administration of the antineoplastic agent, indicating that the extracorporeal circulation treatment after administration of the antineoplastic agent of the present invention is effective.

It is a graph which shows the effect with respect to NK activity at the time of performing extracorporeal circulation treatment using the column which concerns on this invention.

Claims (13)

An immune cell activator obtained by immobilizing lipoteichoic acid on a water-insoluble carrier having an amino group or a functional group capable of covalently bonding to a hydroxyl group. The immune cell activator according to claim 1, wherein the lipoteichoic acid is derived from a Gram-positive bacterium. 2. The immune cell activator according to claim 1, wherein the lipoteichoic acid is derived from hemolytic streptococci. 2. The immune cell activator according to claim 1, wherein the lipoteichoic acid is derived from Bacillus subtilis. The functional group capable of being covalently bonded to the amino group or the hydroxyl group is at least one selected from the group consisting of an active halogen group, an isothiocyanate group, an isocyanate group, an aldehyde group, and a carboxyl group. The immune cell activation material of any one of 1-4. The immune cell activator according to any one of claims 1 to 5, wherein the water-insoluble carrier is an aromatic compound polymer. The immune cell activation material according to any one of claims 1 to 6, wherein the shape is a fiber, a membrane, a hollow fiber, or a granular material. A column packed with the immune cell activation material according to claim 7. The column according to claim 8, which is used for extracorporeal circulation therapy. The method for using a column according to claim 9, wherein the column is used for extracorporeal circulation before, after or simultaneously with the administration of the antineoplastic agent. The method of using a column according to claim 10, wherein the column is used for extracorporeal circulation after administration of an anti-malignant tumor agent. The method of using a column according to claim 10 or 11, wherein the antineoplastic agent is an antimetabolite antineoplastic agent. The method of using a column according to claim 12, wherein the antimetabolite antineoplastic agent is gemcitabine.

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