JP2000217909A - Absorbent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To absorb and remove an oncocyte in body, especially in a blood vessel effectively by preparing an absorbent by dint of fixing ligand having affinity to material which is developed on a surface of the oncocyte with a high polymer. SOLUTION: A material which is developed on a surface of an oncocyte is a molecule having a function to glue cells each other and to be glued with an extracellular matrix or to transmit information of the inside and outside of a cell. The material is composed mainly of protein, polysaccharide and their complex, for example, integrine, selectin CD44, a serial Lex and a serial Lea and so forth is enumerated. An absorbent is prepared by fixing ligand that has affinity for such materials which is developed on a surface of the oncocyte with a high polymer. Ligand may be used if ligand can combine a cell surface material specifically by electrostatic interactions, hydrophobic interaction, van der Waals forces, for example, polypeptide, oligopeptide, sugar and vitamin acid are enumerated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an adsorbent for adsorbing tumor cells in a liquid and an adsorber using the same.

[0002]

BACKGROUND OF THE INVENTION Preventing cancer metastasis is one of the most important tasks in the medical field. Improvements in testing and diagnostic techniques have greatly contributed to the early detection of cancer, but nonetheless, there are many cases where metastasis has already occurred at the time of diagnosis, and the current situation is that the 5-year survival rate of most cancers is low. Cancer metastasis is either spontaneous or artificial, and the former is a case in which cancer cells produce proteases themselves, invade blood vessels, and are transported throughout the bloodstream. The latter is a case in which a cancer tissue or a surrounding cancer cell leaks into a blood vessel or the like by a resection operation in a cancer tissue resection. The presence of cancer cells in the bloodstream is proved by the detection of mRNA in the blood of cancer patients, using messenger RNA (mRNA), which encodes information on proteins contained in the cancer cells, as an indicator. (Mori.Met al. Int J Cancer 1996; 68 (6): 739-74
3, Lemonie. A et al. Ann Sur 1997; 226 (1): 43-50.).
In all cases of metastasis, chemotherapy using an anticancer drug has been conventionally performed. Anticancer drugs have functions such as DNA replication inhibition, protein synthesis inhibition, and metabolism inhibition.However, since they affect not only tumor cells but also normal cells, severe effects such as nephrotoxicity, bone marrow toxicity, vomiting, hair loss, and neuropathy Adverse effects and significantly impaired the patient's Quality of Life.

[0003]

At present, side effects are suppressed by the dose, period of administration, etc. of the anticancer drug.
In particular, it is difficult to control the concentration of the anticancer drug in the local part of the tumor tissue, and no remarkable therapeutic or preventive effect has been obtained. In addition, a method has been devised to remove tumor cells in ascites fluid or bone marrow fluid using activated carbon, a membrane containing cellulose as a main component, or a filter for removing leukocytes, but such a method has been devised for extracorporeal blood circulation. When applied, not only tumor cells but also leukocytes and hematopoietic stem cells are removed at the same time. For cancer patients with reduced immunity and patients in need of hematopoietic stem cell transplantation, the removal of leukocytes and hematopoietic stem cells may promote side effects or reduce the success rate of transplantation.

[0004] In view of the above-mentioned problems of the prior art, the present inventors have studied a method for adsorbing and removing tumor cells leaked into the body, particularly into blood vessels. The present inventors have found that a carrier in which an affinity ligand is immobilized by chemical bonding adsorbs tumor cells, and arrived at the present invention. That is, an object of the present invention is to provide an adsorber that adsorbs and removes tumor cells in the body, especially in blood vessels.

[0005]

The present invention has the following configuration to achieve the above object. "(1) an adsorbent obtained by immobilizing a ligand having an affinity for a substance expressed on the surface of a tumor cell to a polymer compound, and (2) an adsorbent obtained by filling the adsorbent of the above (1). And (3) an extracorporeal circulation system including the adsorber of (2) above, a pump and a catheter for sending blood, and (4) removing the tumor cells in the liquid using the adsorber of (2). how to."

[0006]

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

The substance expressed on the surface of a tumor cell in the present invention is generally a molecule having a function such as adhesion between cells, adhesion to an extracellular matrix, or information transmission inside and outside a cell. Substances having these functions are mainly composed of proteins and polysaccharides, and complexes thereof, but are not particularly limited, for example, integrins, selectins,
Adhesion molecules such as CD44, sialyl Lex, sialyl Lea, growth factor receptors such as insulin-like growth factor I receptor and insulin-like growth factor II receptor, extracellular matrix such as fibronectin and collagen, MUC-
1, mucins such as DF-3, cytokeratin and the like.

[0008] In the present invention, a ligand having an affinity for a substance expressed on the surface of a tumor cell refers to a cell surface substance that is specific for electrostatic interaction, hydrophobic interaction, van der Waals force, or the like. What is necessary is just to be able to couple | bond together, and it does not specifically limit. Those known to be capable of specific binding include monoclonal antibodies, polyclonal antibodies, fragments containing the antigen-binding site of the antibody,
Cell surface receptors, fragments containing the ligand binding site of the receptor, sugar chains, polypeptides, oligopeptides, amino acids, polynucleotides, oligonucleotides, nucleotides, lipids, etc., as well as artificially synthesized organic compounds Ligands are available.
Examples of ligands include polypeptides or oligopeptides such as an antibody such as an anti-integrin antibody, an anti-CD44 antibody, an anti-MUC-1 antibody, an anti-cytokeratin antibody, or a part of an antibody including an antigen recognition site, and an oligo-containing RGD sequence. Examples thereof include peptides, polysaccharides such as hyaluronic acid and phosphomannan, monosaccharides such as mannose-6-phosphate, oligosaccharides such as mannose-6-phosphate pentamer, and vitamin acids such as retinoic acid.

The high molecular compound referred to in the present invention is selected from low-toxic polymers which do not dissolve in contact with body fluids and cause no harm even in contact with blood cell components. Examples thereof include polyurethane, polystyrene, polysulfone and polysulfone. Examples include vinyl chloride, acrylic resin, polyamide resin, vinyl resin, phenoxy resin, urethane resin, fluorine resin, silicon resin, cellulose resin, chitin, chitosan, agarose, and dextran. The polymer material may be composed of one of these polymers alone, or may be composed of a copolymer, a composite or a mixture thereof. In the case of a copolymer, any of a block copolymer, a graft copolymer, and a random copolymer may be used.

In the present invention, the ligand is immobilized by a chemical bond. Examples of the chemical bond include a covalent bond, an ionic bond, and a hydrophobic bond. Among them, a covalent bond is preferable because immobilization is sufficiently possible. . In order to be immobilized by a covalent bond, these polymer materials preferably have an appropriate functional group, for example, an amino group, a hydroxyl group, a carboxyl group, a thiol group, an isocyanate group,
It preferably has a halogen group, a haloacetamidomethyl group and the like. If the number of functional groups is too small, the number of ligands that can be immobilized will be small, so that the adsorption effect will not be exhibited.If the number is too large, excess functional groups that have not reacted with the ligands will unspecifically bind proteins and the like. There is a possibility that adsorption may hinder the adsorption of tumor cells. Therefore, the amount of the functional group is preferably 1 fmol to 10 mol per 1 g of the polymer material, and furthermore
1 pmol to 1 mol is more preferred.

The method of immobilizing the ligand on the polymer compound may be any method, and if there is a reactive functional group, it may be bonded directly using a reactive functional group or via a spacer having an appropriate length. In the case where there is no functional group, bonding may be performed after introducing the functional group. In order to increase the degree of freedom of the ligand, a binding method via a spacer is preferably used. As the spacer, for example, a substance containing polyethylene glycol, polypropylene glycol, polyethylene imine, polymethylene or the like is used. If the amount of immobilized ligand is too small, the adsorption effect tends to be insufficient.If the amount is too large, the ligands may overlap with each other and hinder each other.
1 fmol-10 mol is preferable per 1 g, and 1 pmol-1 mol is more preferable. The ligand to be immobilized is not limited to one type, and a plurality of ligands may be immobilized.

The method for producing the adsorbent of the present invention may be arbitrary,
The adsorbent of the present invention can be obtained by immobilizing the ligand on the polymer compound or by performing the polymerization reaction after immobilizing the ligand on the monomer of the polymer compound in advance. Further, the ligand may be immobilized after the polymer compound is molded, or may be molded after the ligand is immobilized. The carrier surface may be coated with an adsorption material.

The shape when the polymer material of the present invention is packed in a column is not particularly limited. For example, beads, films, fibers or a knitted fabric obtained by knitting the same,
Examples include hollow fibers and gels. When used as an extracorporeal circulation column, beads, fibers, knitted fabrics, and hollow fibers are preferred in that they have as large an area as possible to come into contact with body fluids and can withstand circulation pressure. The adsorption material to be packed in the column is not limited to one kind, and a plurality of ligands in which each ligand is immobilized may be packed. As the liquid passes through the column, it enters the column through the liquid inlet and exits the liquid outlet in direct contact with the adsorbent material. During this time, those having affinity with the ligand are adsorbed.

The adsorber of the present invention can be used for removing cells, especially tumor cells, and is effective for liquids containing tumor cells. For example, a method of removing stored blood containing tumor cells, bone marrow fluid, and lymph fluid by batch-wise removal, or a method of removing tumor cells in circulating blood by embedding in a cancer patient's body by connecting an adsorber to a catheter, There is also a method in which an adsorber is incorporated in an extracorporeal circulation circuit, a pump is connected, blood, ascites, and peritoneal washings are taken out of the body, circulated and removed, and returned to the body. In addition, it has been confirmed at the gene level and the cell level that tumor cells are present in the body fluid of a cancer patient, and a method of removing tumor cells by incorporating an adsorber into a blood circulation circuit is particularly preferable. By reducing the number of tumor cells in the blood before, during and after resection surgery, the efficacy of the anticancer agent can be improved and the probability of metastasis can be significantly reduced. The liquid does not necessarily need to include a body fluid, and for example, tumor cells can be removed from a liquid medium or physiological saline in which various cells including tumor cells are suspended. The extracorporeal circulation circuit includes, for example, a catheter for collecting a fluid containing body fluid from the patient's body, the adsorber of the present invention, a catheter for returning the purified fluid containing fluid to the patient's body, and these devices. It consists of connecting catheters, which are connected in this order to form a circuit. Devices such as a body fluid transport pump, a three-way stopcock, a blood pressure meter, and a blood flow meter may be incorporated on the way. The extracorporeal circulation system is a form of treatment that includes the extracorporeal circulation circuit, transport pump, measurement device, etc. Point to.

Examples are shown below, but the present invention is not limited to these examples.

[0016]

Example 1 Forty polystyrene beads containing 1 nmol of amino groups per bead were mixed with 15 ml of diglycidyl polyethylene glycol.
And stirred at room temperature for 1 day. After washing the beads with methanol, the beads were placed in 15 ml of methanol, 1 ml of ethylenediamine was added, and the mixture was stirred at room temperature for 1 day. After washing the beads with methanol, they were dried (the beads obtained here are hereinafter referred to as beads A). Example 2 20 beads A were mixed with a 0.2 mol / l phosphate buffer solution (pH = 7.0) 10
Add 10 mg of mannose-6-phosphate pentamer and 1.8 mg of tetramethylammonium borohydride, react at 4 ° C overnight, wash with water, dry, and fix mannose-6-phosphate pentamer The modified beads were prepared. Example 3 20 beads A were put in a 0.2 mol / l phosphate buffer solution (pH = 7.0) 10
Then, 2 mg of mannose-6-phosphate and 1.8 mg of tetramethylammonium borohydride were added and reacted at 4 ° C. overnight, washed with water and dried to prepare beads having immobilized mannose-6-phosphate. Example 4 20 beads A were mixed with 0.2 mol / l phosphate buffer solution (pH = 7.0) 10
Then, 500 mg of phosphomannan and 1.8 mg of tetramethylammonium borohydride were added and reacted at 4 ° C. overnight, washed with water and dried to prepare phosphomannan-immobilized beads. Example 5 The ligand-immobilized beads described in Examples 2, 3, and 4 were placed in Dulbecco's modified Eagle's minimum essential medium (D-MEM) in which human breast cancer cell line MCF-7 was suspended, and incubated at 37 ° C. for 1 hour. Shake.
The number of MCF-7 cells not adsorbed to the beads was determined by removing the beads and counting the number of cells in the remaining suspension. As a result, the number of cells in the suspension after the addition of the beads was 43% lower than that before the addition of the beads, 43% for the beads of Example 3, 65% for the beads of Example 3, and 73% for the beads of the Example 4. Was. Example 6 The ligand-immobilized beads described in Example 2 were placed in D-MEM in which human colon cancer cell line Caco-2 was suspended, and shaken at 37 ° C. for 1 hour. The beads were removed, and the number of Caco-2 in the suspension was measured. As a result, the number of cells in the suspension was reduced to 83% of that before the beads were put. Example 7 The ligand-immobilized beads described in Examples 2, 3, and 4 were placed in phosphate-buffered saline (PBS) in which leukocytes collected from heparin-collected human healthy human blood were suspended at 37 ° C. for 1 hour. Shake. As a result of removing the beads and counting the number of leukocytes in the suspension, the number of cells in the suspension did not decrease to almost 100% as compared to before the introduction of the beads. Example 8 One catheter was connected to each side of the inlet and outlet of the column filled with the ligand-immobilized beads described in Example 2. The respective tips of both catheters were placed in D-MEM in which the human breast cancer cell line MCF-7 was suspended, and circulated at 37 ° C. for 1 hour by driving with a rotary pump. MCF- in suspension
As a result of counting the number of cells, the number of cells in the suspension was reduced to 55% as compared with the number before the addition of the beads. Comparative Example 1 20 beads A were mixed with 0.2 mol / l phosphate buffer solution (pH = 7.0) 10
and add 10 μl of 90% acetaldehyde aqueous solution to
The reaction was carried out overnight at ℃, washed with water and dried.

The obtained beads without ligand were placed in D-MEM in which a human breast cancer cell line MCF-7 or a human colon cancer cell line Caco-2 was suspended, and shaken at 37 ° C. for 1 hour. The beads were removed and the number of MCF-7 or Caco-2 in the suspension was measured. As a result, the number of cells in the suspension was hardly reduced to almost 100% of that before the injection of the beads. . Comparative Example 2 PBS in which leukocytes collected from blood of a healthy human subject were obtained by heparin-collecting the ligand-immobilized beads described in Comparative Example 1.
And shaken at 37 ° C. for 1 hour. Remove the beads,
As a result of counting the number of leukocytes in the suspension, the number of cells in the suspension was reduced to 79% as compared to before the beads were put.

[0018]

According to the present invention, cancer cells can be specifically removed from a liquid by passing the liquid containing the cancer cells, and extracorporeal circulation using the present adsorber is carried out before, during and after the operation of cancer resection. This can remove cancer cells scattered in the blood and prevent metastasis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G03G 15/16 103 G03G 15/16 103

Claims (23)

[Claims] 1. An adsorbent in which a ligand having an affinity for a substance expressed on the surface of a tumor cell is immobilized on a polymer compound by chemical bonding. 2. The adsorbent according to claim 1, wherein the ligand having an affinity for the substance expressed on the surface of the tumor cell is at least one selected from a polypeptide, an oligopeptide, a peptide, a sugar and a vitamin acid. 3. The adsorbent according to claim 2, wherein the polypeptide is an antibody or a part thereof. 4. The adsorbent according to claim 2, wherein the saccharide contains a phosphorylated saccharide. 5. The adsorbent according to claim 2, wherein the saccharide contains a mannose pentamer terminated by a phosphorylated saccharide. 6. The adsorbent according to claim 2, wherein said sugar is a phosphorylated sugar. 7. The adsorbent according to claim 4, wherein the phosphorylated sugar is mannose-6-phosphate. 8. The adsorbent according to claim 2, wherein said sugar is phosphomannan. 9. The adsorbent according to claim 2, wherein said vitamin acid is retinoic acid. 10. The method according to claim 1, wherein said polymer compound is a bead.
An adsorbent according to any one of claims 1 to 9. 11. The method according to claim 1, wherein the polymer compound is a fiber.
10. The adsorbent according to any one of 9. 12. The method according to claim 1, wherein said polymer compound is a hollow fiber.
An adsorbent according to any one of claims 1 to 9. 13. The method according to claim 1, wherein the polymer compound is a gel.
10. The adsorbent according to any one of 9. 14. The adsorbent according to claim 1, which adsorbs tumor cells. 15. An adsorber filled with the adsorbent according to claim 1. Description: 16. The adsorber according to claim 15, wherein the tumor cells are specifically removed from the liquid by passing the liquid containing the tumor cells. 17. The adsorber according to claim 16, wherein the liquid is a liquid containing a body fluid. 18. The adsorber according to claim 17, wherein said body fluid is a body fluid selected from blood, lymph, ascites, pleural effusion and bone marrow fluid. 19. The method according to claim 1, wherein said adsorber is a circulation column.
19. The adsorber according to any one of 5 to 18. 20. The adsorber according to claim 19, wherein the circulation column is an extracorporeal circulation column. 21. An extracorporeal circulation system including the adsorber according to claim 15, a pump for sending blood, and a catheter. 22. A tumor cell in a liquid is removed using an adsorber having a built-in adsorbent in which a ligand having an affinity for a substance expressed on the surface of a tumor cell is fixed to a polymer compound by chemical bonding. how to. 23. A method for removing tumor cells in a liquid using the adsorber according to any one of claims 15 to 20.