JPH0767955A - Production inducing method for tumor necrosis factor - Google Patents

Abstract

PURPOSE:To provide the production inducing method for tumor necrosis factor(TNF) excellent in the operability, practicality, effectiveness, and safety. CONSTITUTION:Blood is brought into contact with a polymer material having at least one functional group selected among the hydroxyl group, amide group, and ester group or a material having the cationic functional group, and the production of TNF is induced in this TNF production inducing method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inducing production of TNF by bringing a material and blood into contact with each other, and to obtain blood or blood components (plasma or serum) containing high density of natural TNF. A method of obtaining, relating to blood, blood components and natural TNF thus obtained. In addition, blood, blood components and natural type T thus obtained
The present invention relates to a method of exerting a therapeutic effect on, for example, cancer by returning NF to a patient.

[0002]

As is well known, killer cells, NK cells, LAK cells, activated macrophages, and the like play important roles as antitumor cells that play a role in immune surveillance against malignant tumors in the living body. In addition, interleukins, interferons, TNF, etc. secreted from these cells and lymphocytes are also significantly involved in the antitumor properties of the living body. Therefore, as an immunological treatment method for malignant tumors, it is possible to activate these cells of a patient to effectively induce an antitumor effect, or to induce these cytokines to exert an antitumor effect. Conceivable.
However, it has been reported that the immunocompetence generally decreases with the progression of cancer in cancer patients, and it is considered that the immunocompetence is suppressed in the living body of cancer patients by immunosuppressive factors and the like. ing.

It can be said that it is difficult to effectively induce an antitumor effect in the body of a cancer patient whose immunocompetence is lowered. Therefore, in order to stimulate the immune system in the body of cancer patients and induce antitumor properties, a method of introducing the induced antitumor cells into the body of patients with reduced immunocompetence,
Alternatively, a method of administering a cytokine having an immunostimulatory action into the body is considered as a cancer treatment method.

Recently, recombinant human interleukin-2 was added to the lymphocytes of a cancer patient taken out of the body, cultured, activated to induce LAK cells, and then returned to the body of the cancer patient to exert an antitumor effect. Adoptive immunotherapy and therapeutic methods of administering TNF, which specifically attacks tumor cells, into cancer patients have been tried (Rosenberg, SA, Lotze,
MT, Muul, LM et al .: A Progress report on the tre
atment of 157 patients with advanced cancer using
lymphokine-activated killer cells and interleukin
2 or high-dose interleukin 2 alone.N.Engl.J.Med.
316: 889-897, 1987).

In addition, attempts have been made to induce endogenous TNF by administering bacterial cell components and the like to cancer patients (Haruyuki Ohshima, Genichiro Sogen, Denichi Mizuno: Tumor Necrosis Factor (TN).
F): Application of endogenous TNF production to human cancer and its prospect. B
IOTHERAPY, 1: 145-151,1987, Mikio Kato, Koichi Okada, Genichiro So, Shouchi Takeuchi, Denichi Mizuno: Endogenous and Exogenous TN
F (FET) therapy Clinical results from a multicenter study. BI
OTHERAPY, 5: 473-477, 1991).

[0006] Furthermore, basic research is underway to treat cancer patients by circulating blood through a material on which TNF production-inducing lipopolysaccharide (LPS) is immobilized (Gen Abe: LPS fixation). Anti-tumor effect of derivatized beads, J. Geikai, 92: 627-635, 1991). Similarly, it is also considered that LPS is contacted with blood through a semipermeable membrane or a selectively permeable membrane to return LPS-treated blood to the patient to activate the immune system. 113465).

[0007]

In the adoptive immunotherapy, a large amount of lymphocytes are taken out from a cancer patient, cultured aseptically for a long time in the presence of interleukin 2, and then injected into the cancer patient. Therefore, there are many problems such as that it takes a lot of time and labor, and that the culture takes a long time.

Recently, TN has been obtained by gene recombination.
F can be produced, and clinical tests for cancer treatment using recombinant human TNF are underway (Shigeo Ikeda,
Ishihara K: Phase II clinical trial of genetically engineered human tumor necrosis factor Sertenef (PT-050) for malignant skin tumors. Skin
Cancer, 5: 210-226, 1991). However, although TNF is a cytokine that has both an immunostimulatory effect and an effect of directly killing tumor cells, the effect of recombinant TNF was not as large as expected. It is considered that this is because the recombinant form is not endogenous from the patient itself, so that the effect is small and the side effects are large.

Therefore, bacterial cell components such as BCG and OK-4
Attempts are being made to administer drugs such as 32 to patients to induce endogenous TNF to treat cancer.
However, the induction of TNF and the like by bacterial components causes side effects because the bacterial components undergo various reactions in the body of a cancer patient.

Further, the blood circulation method using the LPS-immobilized material has a problem that LPS is desorbed, migrates into the body, and causes side effects. In general, many immunostimulatory substances with strong immunostimulatory properties are also highly toxic, and if they are wrapped in a semipermeable membrane or immobilized, they must be completely suppressed from leaking into the blood or desorption. Is difficult and cannot guarantee safety.

[0011] Further, TNF is known to exhibit various physiological activities in the living body, and attention is focused on the relationship between the dynamics of TNF in the body and the pathological conditions of various diseases. Therefore, the amount of TNF in blood has been conventionally measured using recombinant TNF as a standard, but it is guaranteed that this recombinant TNF exhibits the same reactivity as natural TNF present in blood. There is no. Therefore, it is desired to use natural TNF as a standard product in order to obtain accurate measurement results.

In order to solve the above-mentioned problems, the present invention provides a method for inducing TNF production which is dramatically improved in operability, practicality, effectiveness and safety as compared with the conventional methods. is there.

[0013]

The method for inducing TNF production according to the present invention comprises a polymer material having one or more functional groups selected from a hydroxyl group, an amide group and an ester group, or a polymer material having a cationic group. It is characterized by inducing the production of TNF by bringing a molecular material and blood into contact with each other.
It is characterized in that they are contacted in the range of 52 ° C.

When leukocytes such as monocytes and neutrophils in blood react with foreign substances, various enzymes and mediators are released. A similar reaction also occurs when these cells come into contact with the material. This occurs when the material is regarded as a foreign substance, and this reaction greatly depends on the nature of the material. Therefore, it is considered that this reaction can be controlled by controlling the properties of the material.

In this reaction, we show that T
Focusing on the production of NF, we have conducted diligent research on the induction of TNF production by contacting various natural and synthetic polymeric materials with blood, and that various polymeric materials induce remarkable production of TNF in blood. I have found In particular, hydroxyl groups,
By contacting blood with a polymer material having an amide group and / or an ester group or a polymer material having a cationic functional group, a remarkable induction of TNF production was observed, whereas polystyrene or polyethylene was used. For polymeric materials such as, polypropylene, polyvinyl chloride,
Almost no induction of TNF production was observed. Polymeric materials having only anionic groups such as sulfonic acid and carboxyl groups have only been observed to induce low levels of TNF production.

Hydroxyl group, amide group, used in the present invention,
Polymer materials having one or more functional groups selected from ester groups include agarose, cellulose, starch, pullulan, dextran, glycergen, mannan, glucomannoglycan, galactomannoglycan, pectin, alginic acid, Natural polysaccharides such as hyaluronic acid, chondroitin sulfate, chitin, chitosan and their carboxymethylation, succinylation, sugar side chain graft, peptide side chain graft, glycolation, acylation,
There are various derivatives modified by amination.

For example, according to the findings obtained by the present inventors, chitin and its N-deacetylated product, chitosan, and chitosan derivatives and alkyl groups in which 1, 2, 3, 4 primary amines are introduced into the amino groups of chitosan. The introduced derivative, the derivative having a sulfone group or a carboxymethyl group introduced into the hydroxyl group, etc. showed a large induction of TNF production upon contact with blood. In addition, agarose and agarose can be used for 2-3
Cross-linked agarose with increased strength by cross-linking dipromopropanol under strong alkaline conditions, and agarose derivatives in which ion exchange groups such as diethylaminoethyl (DEAE) groups are ether-bonded, quaternary amines, etc. The contact of gel beads composed of the modified agarose derivative with blood also showed a remarkable method of inducing TNF production. Further, a remarkable induction of TNF production was also observed by contacting blood with alginic acid gel beads prepared by dissolving sodium alginate or the like in water and bringing them into contact with an aqueous solution containing a polyvalent metal ion.

The polymeric materials having a hydroxyl group, an amide group and an ester group used in the present invention include polyvinyl alcohol, polyhydroxyethyl acrylate, polyhydroxymethyl acrylate, polyphenol, polyvinylpyrrolidone, polyacrylamide, polylysine,
Examples thereof include synthetic polymer materials such as polyvinylpyrrolidone, polyacrylic acid ester, and polymethacrylic acid ester, various derivatives and crosslinked products thereof, and various copolymers of them with vinyl monomers such as styrene and methacrylic acid ester.

For example, according to the knowledge obtained by the present inventors, polyvinyl alcohol gel is obtained by contacting blood with TMF.
It showed a significant induction of production. This polyvinyl alcohol gel introduces crosslinking by adding boric acid, or uses a photocrosslinkable polyvinyl alcohol having a styrylpyridinium group or a styrylquinolinium group which is a photosensitive functional group in a side chain, or as a gelling agent. It can be prepared by adding various organic and inorganic compounds to be referred to, or by repeating the freeze-drying or freeze-thawing of the aqueous solution.

[0020] Further, according to the knowledge obtained by the present inventors, a methacrylic acid ester monomer having the above-mentioned functional group is polymerized and copolymerized with various vinyl monomers to obtain a modified product having a hydroxyl group introduced or an alkyl group. It was also confirmed that TNF was significantly induced in the material into which the phenyl group and the phenyl group were introduced.

The polymeric material having a cationic functional group used in the present invention is a polymeric material having a primary to quaternary amino group, a sulfonium group, a phosphonium group, etc.,
For example, polyvinyl pyridine and its salt, ionene polymer, N-trialkylaminomethyl polystyrene, amino acetalized polyvinyl alcohol, polyvinyl imidazole, polyethylene imine, polydialkyl diallyl ammonium salt, polydialkyl diallyl ammonium salt-SO ₂ copolymer, Examples thereof include polyvinylbenzylsulfonium salt and polyvinylbenzylphosphonium salt.

The polymeric material having the above-mentioned cationic functional group can be introduced into the above-mentioned cationic functional group or synthetic functional polymers such as polystyrene by using various chemical modification methods. It can be obtained by a copolymerization or a crosslinking reaction between the vinyl monomers. For example, by copolymerizing styrene and divinylbenzene, Friedil-
It is possible to introduce a cationic functional group into polystyrene by introducing a chloromethyl group into a benzene nucleus through a Krafts reaction, treating the chloromethyl group with an amine to aminate, and then performing alkyl substitution. it can.

As another method, for example, a compound having a chloromethyl group and an oxirane ring in the molecule such as epichlorohydrin is reacted with an imidazole to synthesize a modified imidazole, which is then treated with a resin having a polyfunctional epoxy compound. It can also be obtained by converting. Further, regarding the polymer material having a cationic functional group,
Various other synthetic methods are possible, but the present invention is not limited to these methods.

According to the knowledge obtained by the present inventors, a polystyrene modified product in which a trimethylammonium group having a quaternary ammonium trialkyl-substituted nitrogen atom or a dimethylethanolammonium group which is a dialkylethanol is introduced as a cationic group is not known. Compared with the material composed of modified polystyrene or polystyrene modified with anionic group, a significantly larger induction of TNF production was shown. In addition, a large induction of TNF production was confirmed even with a cationic group having a primary or secondary amino group or a tertiary amino group. On the other hand, in the case of polystyrene derivatives in which a sulfonic acid group, which is an anionic group, is introduced, almost no
No induction of production was observed.

The polymethacrylic acid ester-based material obtained by copolymerization of a methacrylic acid ester monomer having a cationic group such as a dimethylethanolammonium group, a diethylamino group, and a primary amino group at the terminal has a remarkable TNF Induction of production was shown. On the other hand, almost no induction of TNF production was observed in the methacrylic acid ester-based material having a carboxyl group as an anionic group.

Further, chitosan which is an N-deacetylated product of chitin and a chitosan derivative obtained by introducing a primary to quaternary amine into the amino group of chitosan, diethylaminoethyl (D
As described above, the agarose derivative in which an ion exchange group such as an EAE) group is ether-bonded, and the agarose derivative modified with a quaternary amine show a particularly high TNF production induction.

As the shape of various TNF production inducing materials used in the present invention, any known shape such as particle shape, fiber shape, hollow fiber shape and film shape can be used.
For example, divinylbenzene and a radical initiator benzoyl peroxide are added to a styrene monomer to prepare 6 in water.
If the suspension and stirring are continued at 0 ° C. for 5 hours, spherical particles of a styrene / divinylbenzene copolymer can be easily obtained. The particle size can be controlled by the stirring speed, the type and concentration of the stabilizer added to water, the volume ratio of the monomer to water, and the like. Further, it is also easy to make it porous by carrying out suspension polymerization of a mixed liquid of a diluent and a monomer. Further, when the polymerization of various methacrylic acid ester monomers is carried out by suspension polymerization in a good solvent for the monomers and a poor solvent for the polymers, spherical particles of polymethacrylic acid ester can be easily obtained. The particle shape can be controlled by the stirring speed, the type and concentration of the stabilizer added, and the like. The spherical particles thus synthesized are subjected to the chemical modification reaction as described above to obtain various TNF production inducing materials.

Regarding the method of contacting blood with the above-mentioned material, any method can be used as long as blood can be sufficiently contacted with the above-mentioned material. For example, a method in which the above fibrous material is packed in a column and blood is circulated in the column, or a method in which a bead-shaped material having a particle size of 50 μm to 5 mm is packed in the column and blood is circulated can be used.
Further, the material may be brought into contact with blood by suspending the material having various shapes in the blood.

The temperature at which the above material is brought into contact with blood is
The range of 15 ° C to 52 ° C is preferable in order to enhance the induction of TNF production. According to the knowledge obtained by the present inventors, when the contact temperature is lower than 15 ° C., the T
No induction of NF production was observed. Also, the contact temperature is 5
When the temperature was higher than 2 ° C, denaturation of plasma protein, remarkable hemolysis, and leukocyte breakdown occurred, and the induction of TNF production was drastically reduced.

In the present invention, the contact between the material and blood causes the interaction between the material and cells,
Induction of TNF production is performed. The TNF-producing cells are not limited to cells in peripheral blood, but also include cells obtained from lymph vessels, lymph nodes, pancreas and the like. The blood is rich in these cells that produce TNF. Although these cells in the blood act on the above-mentioned material to induce the production of TNF, even if they do not act directly, the TNF is mediated by another factor induced by the action of the above-mentioned material and some factor in the blood.
May be induced.

Using the above materials, TN was obtained from the blood of a cancer patient.
By inducing the production of F, it becomes possible to use it for the treatment of cancer or the like and to obtain natural TNF. In the treatment, endogenous TN derived from a cancer patient is obtained by returning TNF-induced blood to the cancer patient using an extracorporeal circulation system or the like.
F exerts an antitumor effect in the patient. It is also possible to separate plasma or the like from the contacted blood and administer the plasma or the like of the cancer patient's own TNF-producing inducer systemically or locally to the tumor in a timely manner.

These treatment methods will be described in more detail. First, for treatment of cancer patients and the like, extracorporeal circulation treatment of blood using a column filled with these materials can be performed.
Blood of a cancer patient or the like is guided to the extracorporeal circulation system using a blood tube or the like, and introduced into a column packed with a TNF-inducing material in the form of fibers or beads. In the column, the contact of blood with the material induces endogenous TNF in the blood. By returning blood containing high-density endogenous TNF to the body of a cancer patient or the like again, the patient-derived T
NF will be administered. In addition, the blood of a patient who has been bled in advance is put in a blood bag or the like filled with the above TNF production inducing material, TNF production is induced therein, and plasma containing patient-derived endogenous TNF at a high density is collected. To do. This plasma or the like is frozen and stored, and if necessary, administered to a patient, effective treatment can be performed. For example, when a tumor is surgically excised, its frozen TNF-producing plasma can be administered systemically or locally to the tumor to prevent the tumor from growing or migrating.

Further, TNF can be easily induced from the blood of a patient by using various TNF production-inducing materials, and the TNF-producing ability of the patient can be detected by measuring the degree of the amount of induction. In other words, the patient's own T
The ability to produce NF can be easily examined. This can be an effective immunological parameter that reflects the patient's condition.

On the other hand, TNF in blood is measured by the conventional method.
Recombinant TN as standard when measuring concentration
Although F is used, plasma containing naturally-occurring TNF at high density, naturally-occurring TNF, TNF derived from the patient himself, and the like are extremely reliable standard products. Therefore, TNF produced by contacting various TNF production-inducing materials with blood
Can be used as a highly reliable standard product, and such TNF can be easily obtained.

[0035]

In the present invention, a polymer material having a hydroxyl group, an amide group, an ester group or a polymer material having a cationic functional group is brought into contact with blood. Therefore, as shown in Examples described later, Therefore, the interaction between TNF-producing cells in the blood and the material is promoted, or the material interacts with some factor,
The production of TNF can be efficiently induced by another factor induced thereby.

[0036]

EXAMPLES The present invention will be described in detail below with reference to examples and comparative examples of the present invention, but the present invention is not limited to the following examples.

(T with chitosan or a chitosan derivative
Induction of NF production) Example 1 Chitosan gel particles Chitosan basic AL-03 in which amino group of chitosan remains as it is
(Fuji Spinning Co., Ltd. product name) was put in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) in a bulk volume of 1 ml.
To this, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred. Then, it was centrifuged at 500 rpm for 5 minutes, and after centrifugation, the supernatant was aspirated and discarded. Further, similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added, and the mixture was stirred, centrifuged at 500 rpm for 5 minutes, and then the supernatant was aspirated and discarded. This washing operation was repeated 3 times and left overnight at 4 ° C. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.

A 2 ml tube (eppendorf), which was sterilized and filled with physiological saline for injection, was filled with the chitosan gel particles washed as described above in a bulk volume of 50.
It was filled with 0 μl and used as Example 1.

Example 2 Chitopearl basic AL-03 was prepared by using chitopea in which the amino group of chitosan was acetylated.
A chitosan derivative-filled tube of Example 2 was obtained in the same manner as in Example 1 except that the tube was changed to rl basic BL-03 (manufactured by Fuji Spinning Co., Ltd.).

Example 3 Chitopearl basic AL-03 was prepared by introducing a primary amine into the amino group of chitosan via an aromatic alkyl group.
A chitosan derivative-filled tube of Example 3 was obtained in the same manner as in Example 1 except that the tube was changed to 3 (manufactured by Fuji Spinning Co., Ltd.).

Example 4 Chitopearl basic AL-03 was prepared by introducing a primary amine into the amino group of chitosan via a linear alkyl group to obtain Chitopearl BCW-3003.
A chitosan derivative-filled tube of Example 4 was obtained in the same manner as in Example 1 except that the tube was changed to (Fuji Spinning Co., Ltd.).

Example 5 Chitopearl basic AL-03 was prepared by using Chitosan in which a tertiary amine was introduced into the amino group of chitosan.
A chitosan derivative-filled tube of Example 5 was obtained in the same manner as in Example 1 except that it was changed to Pearl BCW-2603 (manufactured by Fuji Spinning Co., Ltd.).

Example 6 Chitopearl basic AL-03 was prepared by using Chitosan in which a quaternary amine was introduced into the amino group of chitosan.
A chitosan derivative-filled tube of Example 6 was obtained in the same manner as in Example 1 except that it was changed to Pearl BCW-2503 (manufactured by Fuji Spinning Co., Ltd.).

Example 7 Chitopearl basic AL-03 was prepared by using Chitopearl CM-03 (manufactured by Fuji Spinning Co., Ltd.) in which a carboxymethyl group was introduced into the hydroxyl group at the 6-position of chitosan.
Example 7 is the same as Example 1 except that
A chitosan derivative-filled tube was obtained.

Example 8 Chitopearl basic AL-03 was prepared by introducing a sulfo group into the hydroxyl group at the 6-position of chitosan.
A chitosan derivative-filled tube of Example 8 was obtained in the same manner as in Example 1 except that it was changed to topear SU-03 (manufactured by Fuji Spinning Co., Ltd.).

Example 9 5 g of powder of CHITOSAN 10B (trade name of Higeta Soy Sauce) was added to 30 ml of physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.).
And was centrifuged at 1500 rpm for 1 minute under high speed. After centrifugation, the supernatant was aspirated and discarded, and similarly, 30 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to suspend the solution.
Centrifuge at 0 rpm for 1 minute and then aspirate the supernatant and discard. This washing operation was repeated 3 times and left overnight at 4 ° C.
After that, the same washing operation as above was performed 5 times, and finally the physiological saline was removed as much as possible.

500 ml of the bulk volume of the chitosan powder washed as described above was placed in a 2 ml tube (eppendorf) which was sterilized and filled with physiological saline for injection.
It was filled with μl and used as Example 9.

To each of the tubes obtained in Examples 1 to 9, 1.6 ml of heparin-collected healthy human fresh blood was added, and the mixture was attached to a rotating disk and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm.

The blood after mixing was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a TNF monoclonal antibody by an immunoenzymatic antibody method (Quantitative manufactured by R & D System).
ikineTNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after blood collection, blood was centrifuged to collect plasma, and the concentration of TNF in plasma was measured in the same manner. The results are shown in Table 1.

[0051]

[Table 1]

(Induction of TNF Production by Agarose and Agarose Derivatives) Example 10 Agarose (manufactured by Nacalai Chemical Co., Inc., special reagent for electrophoresis GP-36) was dissolved in distilled water at a concentration of 5% by weight to prepare 1
Autoclave was performed at 21 ° C. for 20 minutes. This solution was kept warm at 60 ° C. and dropped into cold distilled water (4 ° C.) using a microsyringe to prepare agarose gel beads (particle diameter 5 mm).

The beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection 2
It was filled in a ml tube (eppendorf). The filling amount was 20 agarose gel beads.

Example 11 Sepharose 2B consisting of about 2% by weight agarose
(Pharmacia LKB Biotechnolgy) 3 ml suspension,
It was put in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.). This is centrifuged at 1000 rpm for 5 minutes, the supernatant is aspirated and discarded, and physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.)
Was added and stirred, the mixture was centrifuged under the same conditions, and the supernatant was aspirated and discarded. This washing operation was repeated 3 times and left overnight at 4 ° C.

500 μl (bulk volume) of this gel carrier was added to a polypropylene tube for 2 ml (ep) as in Example 1.
(Pendorf) and washed with physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.).

Example 12 Sepharos consisting of cross-linked agarose at a concentration of about 6% by weight
Beads were prepared in the same manner as in Example 11 except that eCL-6B (Pharmacia LKB Biotechnolgy) was used.

Example 13 DEAE S prepared by introducing diethylaminoethyl (DEAE) group through ether bond into cross-linked agarose having a concentration of about 6% by weight.
Beads were prepared in the same manner as in Example 11 except that epharose CL-6B (Pharmacia LKB Biotechnolgy) was used.

Example 14 Phenyl Sepharose CL-4B (P was prepared by introducing a phenyl group into a crosslinked agarose having a concentration of about 4% by weight through an ether bond.
harmacia LKB Biotechnolgy)
Beads were obtained in the same manner as in Example 11.

Example 15 Q Sepharose FF (Pharmacia LKB Biotechnology) prepared by introducing a quaternary amine into cross-linked agarose having a concentration of about 6% by weight.
Beads were obtained in the same manner as in Example 11 except that the same was used.

1.6 ml of heparinized fresh blood of a healthy person was added to a tube filled with each bead of Examples 10 to 15 and attached to a rotating disk, and the rotation speed was 26 at 37 ° C. for 2 hours.
Mix by inverting at rpm.

The blood after mixing was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a TNF monoclonal antibody by an immunoenzymatic antibody method (R & D System Quant.
ikineTNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after the blood collection, blood was centrifuged to collect plasma, and the concentration of TNF in plasma was measured in the same manner. The results are shown in Table 2.

[0063]

[Table 2]

(Induction of TNF Production by Alginic Acid Gel) Example 16 2% by weight of sodium alginate (AL-1, medium viscosity type, manufactured by Nitta Gelatin Co., Ltd.) was suspended in physiological saline and heated at 121 ° C. in an autoclave. By performing the treatment for 20 minutes, the sodium alginate was dissolved simultaneously with the heat sterilization. Particle size of about 2.5 mm by dropping this into sterilized 1.5 wt% calcium chloride solution and gelling.
Calcium alginate gel beads were prepared. The gel beads were placed in a 15 ml polypyroprene tube (manufactured by Iwaki Glass Co., Ltd.) in a bulk volume of 1 ml. To this, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred. Centrifuge at 500 rpm for 1 minute, aspirate and discard the supernatant, and inject saline (Otsuka Pharmaceutical Co., Ltd.) 1
2 ml was added, stirred, centrifuged, and the supernatant was aspirated and discarded. This washing operation was performed 3 times and left overnight at 4 ° C.
After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.

70 gel beads were filled in a 2 ml tube (manufactured by Eppendorf) that had been filled with physiological saline for injection and sterilized and washed.

Example 17 A bead-filled tube was obtained in the same manner as in Example 16 except that 2% by weight of sodium alginate was changed to a concentration of 5% by weight.

Example 18 A bead-filled tube was obtained in the same manner as in Example 16 except that 2% by weight of sodium alginate was changed to a concentration of 10% by weight.

Example 19 A bead-filled tube was obtained in the same manner as in Example 16 except that the 1.5 wt% calcium chloride solution was changed to a concentration of 3 wt%.

Example 20 A bead-filled tube was obtained in the same manner as in Example 16 except that the 1.5 wt% calcium chloride solution was changed to the 1.5 wt% barium chloride solution.

Example 21 All procedures were carried out except that the 1.5 wt% calcium chloride solution was changed to a 1.5 wt% barium chloride solution and the 5 wt% sodium alginate was changed to a concentration of 3 wt%. The beads-filled tube was obtained in the same manner as in Example 16.

Example 22 Sodium alginate was replaced with sodium alginate (100-
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 150 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

Example 23 Sodium alginate (300-300 g
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 400 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

Example 24 Sodium alginate was added to sodium alginate (500-
A bead-filled tube was obtained in the same manner as in Example 16 except that it was changed to 600 cps, manufactured by Wako Pure Chemical Industries, Ltd.).

To a tube filled with each bead of Examples 16 to 24 was added 1.6 ml of heparinized fresh blood from a healthy person, and the tube was attached to a rotating disk and rotated at 37 ° C. for 2 hours at a rotation speed of 2
Mix by inverting at 6 rpm.

The blood after mixing was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a TNF monoclonal antibody by an immunoenzymatic antibody method (Quantum manufactured by R & D System).
ikineTNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after the blood collection, blood was centrifuged to collect plasma, and the concentration of TNF in plasma was measured in the same manner. The results are shown in Table 3.

[0077]

[Table 3]

(Polyvinyl alcohol gel TNF
Induction of Production Example 25 A 10% by weight aqueous solution of polyvinyl alcohol gel (manufactured by Kishida Chemical Co., Ltd.) having a polymerization degree of about 1400 and a saponification degree of 99 mol% was prepared. This aqueous solution was defined as R = [polyvinyl alcohol monomer unit] / [metal ion],
Fe ³⁺ is contained so that R = 10. This viscous solution was added dropwise to a sterilized 1M NaOH aqueous solution using a syringe, and left for about 30 minutes. As a result, polyvinyl alcohol gel beads having a particle size of about 2.5 mm were prepared. [H. Yokoi: Complex gel of PVA and PAA, polymer processing, Vol. 40, No. 11, 991].

The gel beads were placed in a polypropylene tube for 15 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 1 ml. 12m of physiological saline for injection (made by Otsuka Pharmaceutical Co., Ltd.)
1 and added and stirred lightly. Then, the mixture was centrifuged at 500 rpm for 1 minute, and the supernatant was aspirated and discarded. Similarly, 12 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and stirred. Threw away. This washing operation was repeated 3 times and left overnight at 4 ° C. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.

70 pieces of the obtained gel beads were filled in a 2 ml tube (manufactured by Eppendorf) that had been filled with physiological saline for injection and sterilized and washed.

Example 26 The same procedure as in Example 25 was carried out except that the polyvinyl alcohol used was changed to a polyvinyl alcohol (manufactured by Kishida Chemical Co., Ltd.) having a heavy: about 2000 and a saponification degree: 98.5-99.4 mol%. A polyvinyl alcohol gel-filled tube of Example 26 was obtained.

[0082] In the same manner as all except for changing the concentration of the polyvinyl alcohol used in Example 27 to 15% by weight Example 25, to obtain a polyvinyl alcohol gel-filled tube of Example 27.

[0083] In the same manner as in Example 26 except for changing the concentration of the polyvinyl alcohol used in Example 28 to 15 wt%, to obtain a polyvinyl alcohol gel-filled tube of Example 28.

[0084] The quantity of metal ions used in Example 29 in the same manner as in Example 25 except for changing such that R = 20, to obtain a polyvinyl alcohol gel-filled tube of Example 29.

[0085] The quantity of metal ions used in Example 30 in the same manner as in Example 26 except for changing such that R = 20, to obtain a polyvinyl alcohol gel-filled tube of Example 30.

[0086] In the same manner as all except that the quantity of metal ions used in Example 31 was changed to Cu ²⁺ Example 25, to obtain a polyvinyl alcohol gel-filled tube of Example 31.

[0087] In the same manner as in Example 26 except that the quantity of metal ions used in Example 32 was changed to Cu ^2+, was obtained polyvinyl alcohol gel-filled tube of Example 32.

Example 33 Photocrosslinkable polyvinyl alcohol (degree of polymerization: 1700, degree of saponification: 88 mol% and ratio of styrylpyridinium substituents: 1.3%) was added to physiological saline for injection to be 5% by weight. In addition, 121 ℃, 20 by autoclave
The photocrosslinkable polyvinyl alcohol was dissolved at the same time as the heat sterilization by treating with minutes to obtain a photocrosslinkable polyvinyl alcohol solution.

The obtained solution was added to liquid paraffin and stirred sufficiently to suspend the solution. After that, using a slide projector equipped with a 300 W halogen lamp, by irradiating light while stirring for 30 minutes,
Photo-crosslinkable polyvinyl alcohol gelled, particle size 2.5
mm gel beads were prepared. The gel beads were thoroughly washed with physiological saline for injection to remove the attached liquid paraffin.

A polyvinyl alcohol gel-filled tube of Example 33 was obtained in the same manner as in Example 25 except for the above-mentioned method for producing gel beads.

Example 34 The photocrosslinkable polyvinyl alcohol concentration used was 10% by weight.
Except that it was changed to
A polyvinyl alcohol gel-filled tube of Example 34 was obtained.

Example 35 The photocrosslinkable polyvinyl alcohol concentration used was 15% by weight.
Except that it was changed to
A polyvinyl alcohol gel-filled tube of Example 35 was obtained.

To each of the tubes obtained in Examples 25 to 35, 1.6 ml of heparin-collected fresh blood of a healthy person was added and attached to a rotating disk, and mixed by inversion at 37 ° C. for 2 hours at a rotation speed of 26 rpm.

The blood after mixing was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using an immunoenzymatic antibody method (R & D System Quant) using a TNF monoclonal antibody.
ikineTNF-α). The detection limit concentration of this measuring method was 25 pg / ml. In addition, blood immediately after blood collection is centrifuged to collect plasma, and TNF in the plasma is collected.
Was similarly measured.

The results are shown in Table 4.

[0096]

[Table 4]

(Induction of TNF Production by Modified Polystyrene) Example 36 A styrene / divinylbenzene copolymer having a trimethylammonium group which is a quaternary ammonium group, D
iaion SA11A (manufactured by Mitsubishi Kasei) was put in a 50 ml polypropylene tube (manufactured by Iwaki Glass Co., Ltd.) in a bulk volume of 3 ml. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was aspirated and removed. This 3
After repeating the procedure, 40 ml of methanol was added in the same manner and the mixture was allowed to stand at room temperature overnight.

Next, the methanol was removed by suction, and the same washing with methanol was carried out twice. To this, 40 ml of sterilized distilled water was added and gently stirred. The mixture was centrifuged at 500 rpm for 1 minute, the upper portion was removed by suction, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours.

Next, the same washing operation was performed three times, and finally distilled water was removed as much as possible. To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred.
Centrifuge at 500 rpm for 1 minute, aspirate the upper part,
Similarly, physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was washed three times, and finally 40 ml of physiological saline for injection was added, and the mixture was allowed to stand overnight at room temperature. After that, the same washing operation was performed 5 times, and finally the physiological saline was removed as much as possible.

Into a 2 ml tube (manufactured by Eppendorf) which had been filled with physiological saline for injection and sterilized and washed, 500 μl of the polystyrene particles was filled in a bulk volume.

The polystyrene particles used in Example 37 had a quaternary amimonium group, dimethylethanolammonium group, and Diaio.
n SA21A (manufactured by Mitsubishi Kasei Co., Ltd.) was carried out in the same manner as in Example 36 except that the same was used.

The polystyrene particles used in Example 38 were mixed with primary and secondary amino groups [-CH
₂ NH (CH ₂ CH ₂ NH) _n H (n = 1 to 3)], and was changed to Diaion WA21 (manufactured by Mitsubishi Kasei Co.).

The polystyrene particles used in Example 39 were treated with a tertiary amino group [-(C
H ₂ ) _n N (CH ₃ ) ₂ (n = 1 to 3)], and
The same procedure as in Example 36 was performed except that the aion WA30 (manufactured by Mitsubishi Kasei Co., Ltd.) was used.

Comparative Example 1 The same procedure as in Example 36 was carried out except that the polystyrene particles used in Comparative Example 1 were changed to techpolymer: SB-100S (manufactured by Sekisui Plastics Co., Ltd.) which was an unmodified polystyrene particle.

Comparative Example 2 The polystyrene particles used were Diaion S, a styrene / divinylbenzene copolymer having sulfonic acid groups.
The same procedure as in Example 36 was performed except that K1B (manufactured by Mitsubishi Kasei Co., Ltd.) was used.

To the tubes of Examples 36 to 39 and Comparative Examples 1 and 2, 1.6 ml of heparinized fresh blood of a healthy person was added and attached to a rotating disc, and the rotation speed was 26 at 37 ° C. for 2 hours.
Mix by inverting at rpm.

The mixed blood was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a monoclonal antibody by an immunoenzymatic antibody method (R & D System Qu.
Antikine TNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after blood collection, blood was centrifuged to collect plasma, and the TNF concentration in plasma was measured in the same manner. The results are shown in Table 5.

[0109]

[Table 5]

(Induction of TNF Production by Polymethacrylate Derivative) Example 40 SEPABEADS, which is a polymethacrylate ester material modified with dimethylethanolamine
FP-QA13 (manufactured by Mitsubishi Kasei) in polypropylene tube for 50 ml (manufactured by Iwaki Glass Co., Ltd.) with a bulk volume of 3 ml
I put it in. To this, 40 ml of methanol (liquid chromatogram grade manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently. After standing, the supernatant was aspirated and removed. After this was repeated 3 times, 40 ml of methanol was added in the same manner, and the mixture was allowed to stand overnight at room temperature.

Thereafter, the methanol was removed by suction, and the same washing with methanol was carried out twice. To this, 40 ml of sterilized distilled water was added and gently stirred. 500 rpm
The mixture was centrifuged at 1 minute for 1 minute, the supernatant was aspirated and removed, and similarly washed with sterilized distilled water three times. Then, 40 ml of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 3 hours.

Next, the same washing operation was performed three times, and finally distilled water was removed as much as possible. To this, 40 ml of physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added and gently stirred.
Centrifuge at 500 rpm for 1 minute, remove the supernatant by aspiration,
Similarly, physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was washed three times, and finally 40 ml of physiological saline for injection was added, and the mixture was allowed to stand overnight at room temperature. After that, the same washing device was performed 5 times, and finally the physiological saline was removed as much as possible.

Particles of the polymethacrylate ester material were added to a 2 ml tube (manufactured by Eppendorf Co., Ltd.) filled with physiological saline for injection and sterilized and washed to have a bulk volume of 5
00 μl was filled.

Example 41 The procedure of Example 40 was repeated except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-DA13 (manufactured by Mitsubishi Kasei) having a diethylamino group.

The polymethacrylic acid ester material used in Example 42 was prepared by using SEPABEADS FP-HA13 having a primary amino group at the terminal.
The same procedure as in Example 40 was carried out except that the product was manufactured by Mitsubishi Kasei.

The polymethacrylic acid ester material used in Example 43 was prepared by using SEPABEA SFP-HG13 (manufactured by Mitsubishi Kasei) having a hydroxyl group.
Example 40 was performed in the same manner as in Example 40 except that

Example 44 The polymethacrylic acid ester-based material used was the SEPABEADS FP-1 having a hydrophobic group [-OC ₆ H _5].
Example 40 except that the number was changed to 3 (manufactured by Mitsubishi Kasei)
I went in the same way.

Comparative Example 3 The procedure of Example 40 was repeated except that the polymethacrylic acid ester material used was changed to SEPABEADS FP-CM13 (manufactured by Mitsubishi Kasei) having a carboxyl group.

To each tube of Examples 40 to 44 and Comparative Example 3, 1.6 ml of heparin-collected fresh blood from a healthy person was added and attached to a rotating disc, and the rotation speed was 26 r at 37 ° C. for 2 hours.
Mix by tumbling at pm.

After mixing, the blood was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a monoclonal antibody by an immunoenzymatic antibody method (R & D System Qu.
Antikine TNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after blood collection, blood was centrifuged to collect plasma, and the concentration of TNF in plasma was measured in the same manner. The results are shown in Table 6.

[0122]

[Table 6]

(Induction of TNF Production by Hydrophobic Material) Comparative Example 4 2 ml tube (manufactured by Eppendorf) washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.).

Comparative Example 5 Polyethylene beads (particle size 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection to 2 m.
1 tube (made by Eppendorf) was filled. The filling amount was 70 beads.

Comparative Example 6 Polystyrene beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection to 2 m.
1 tube (made by Eppendorf) was filled. The filling amount was 70 beads.

Comparative Example 7 Polyvinyl chloride beads (particle size 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection 2
It was filled in a ml tube (made by Eppendorf). The filling amount was 70 beads.

Comparative Example 8 Polypropylene beads (particle diameter 2.5 mm) were produced by injection molding. The beads were washed with methanol and dried. Next, the beads were washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) and then with physiological saline for injection 2
It was filled in a ml tube (made by Eppendorf). The filling amount was 70 beads.

Comparative Example 9 Polytetrafluoroethylene propylene copolymer beads (particle size 2.5 mm) were prepared by injection molding. Next, the beads were washed with methanol and dried. Next, after washing the beads with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.),
A 2 ml tube (manufactured by Eppendorf) similarly washed with physiological saline for injection was filled. The filling amount was 70 beads.

To each tube of Comparative Examples 4-9, 1.6 ml of heparin-collected healthy human fresh blood was added and attached to a rotating disk, and mixed by inverting at 37 ° C. for 2 hours at a rotation speed of 26 rpm.

The blood after mixing was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a monoclonal antibody by an immunoenzymatic antibody method (R & D System Qu.
Antikine TNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after blood collection, blood was centrifuged to collect plasma, and the TNF concentration in plasma was measured in the same manner. The results are shown in Table 7.

[0132]

[Table 7]

(Effect of Contact Temperature on TNF Production Induction) Example 45 Chitosan Gel Particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at a rotation speed of 26 rpm for 2 hours at 15 ° C.

Example 46 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at 25 ° C. for 2 hours at a rotation speed of 26 rpm.

Example 47 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at 47 ° C. for 2 hours at a rotation speed of 26 rpm.

Example 48 Chitosan gel particles Chitopearl as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at 52 ° C. for 2 hours at a rotation speed of 26 rpm.

Comparative Example 10 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at 10 ° C. for 2 hours at a rotation speed of 26 rpm.

Comparative Example 11 Chitosan gel particles Chitopear as in Example 1
1.6 ml of heparin-collected healthy human fresh blood was added to a polypropylene tube filled with 1 basic AL-03 (trade name, manufactured by Fuji Spinning Co., Ltd.), and attached to a rotary disc.
The mixture was mixed by inversion at 60 ° C. for 2 hours at a rotation speed of 26 rpm.

After mixing, each blood was centrifuged to collect plasma, and the concentration of TNF in the plasma was measured using a monoclonal antibody by an immunoenzymatic antibody method (R & D System Qu.
Antikine TNF-α). The detection limit concentration of this measuring method was 25 pg / ml.

Immediately after blood collection, blood was centrifuged to collect plasma, and the concentration of TNF in plasma was measured in the same manner. The results are shown in Table 8.

[0141]

[Table 8]

The TNF concentration in plasma immediately after blood collection was below the measurement limit, and in the case where a 2 ml tube washed with physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) was just filled with blood (Comparative Example 4), Medium TNF concentration The concentration was 25 pg / ml or less. From the results of Tables 1 to 7, hydroxyl group, amide group,
It is clear that the polymeric material having an ester group and the polymeric material having a cationic functional group induce a particularly high TNF production upon contact with blood.

On the other hand, hydrophobic polymer materials such as unmodified polystyrene, polyethylene and polyvinyl chloride and polymer materials having only anionic functional groups hardly induced TNF production.

As is clear from the results in Table 8, the contact temperature between blood and the above TNF-inducing material was 15 ° C to 52 ° C.
Induction of TNF production was observed in the range of ° C. Also, 15
No induction of TNF production was observed at temperatures below 0 ° C and above 52 ° C.

[0145]

INDUSTRIAL APPLICABILITY According to the present invention, TNF production can be achieved by contacting blood with a polymeric material having a hydroxyl group, an amide group and / or an ester group or a polymeric material having a cationic functional group outside the body. It is possible to easily obtain natural TNF or endogenous TNF derived from a cancer patient by induction, and to provide a new method for the treatment of cancer and the immunological diagnosis for grasping the pathological condition.

Further, unlike the conventional physiologically active substance-immobilized material, there is no detachment of the immobilized physiologically active substance or the like, so that it can be safely applied to the human body.

 ─────────────────────────────────────────────────── ─── Continuation of front page (31) Priority claim number Japanese Patent Application No. 5-114576 (32) Priority date 5 (1993) May 17 (33) Priority claim country Japan (JP)

Claims (3)

[Claims] 1. TNF (TNF: TNF: TNF: TNF:
Tumor Necrosis Factor) production is induced. 2. A method for inducing TNF production, which comprises inducing TNF production by bringing a polymeric material having a cationic functional group into contact with blood. 3. A method for inducing TNF production, which comprises contacting the TNF production inducing material according to claim 1 with blood in the range of 15 ° C. to 52 ° C.