JP2021063023A - Pharmaceutical composition for preventing or reducing resistance to anticancer agent - Google Patents

Abstract

To provide a pharmaceutical composition that has no toxicity, does not affect the metabolism of chemotherapeutics, and prevents or reduces resistance to an anticancer agent, particularly multiple drug resistance (MDR).SOLUTION: A pharmaceutical composition contains nanoparticles comprising biocompatible materials with an average particle size of 300 nm or less. Nanoparticles (TiO2-PEG nanoparticles) comprising complexes of polyethylene glycol (PEG) and TiO2 can cause the migration of P-glycoprotein in a HepG2 cell strain from a cytomembrane to cytoplasm, or reduction in the cytomembrane expression of the P-glycoprotein.SELECTED DRAWING: None

Description

The present invention relates to a pharmaceutical composition that prevents or reduces resistance to an anticancer agent, and more specifically, a medicament that suppresses excretion of an anticancer agent from cancer or tumor cells to prevent or reduce the resistance of cancer or tumor cells to the anticancer agent. Regarding the composition.

Chemotherapy is the primary choice for cancer treatment, and numerous anti-cancer agents have been developed to date, some of which are widely used clinically.
Cancer or tumor cells are characterized by abnormal proliferation that repeats cell division in a short period of time, and most chemotherapeutic agents promote or suppress the death of cancer cells through inhibition of cell division of the cancer cells. Such chemotherapy has the problem of low specificity for cancer cells, large side effects, and thus dose limitation. Chemotherapy also has another major problem, multidrug resistance (MDR), and it is known that many types of cancer or tumor cells acquire resistance to a wide range of structurally different antineoplastic agents. (Non-Patent Document 1). The mechanism of cancer MDR mainly depends on the expression of an energy-dependent efflux pump called an adenosine triphosphate (ATP) -binding cassette (ABC) transporter (Non-Patent Document 2). P-glycoprotein (also called P-glycoprotein, multidrug-resistant protein 1, MDR1, or ABCB1) is one of the most important members of these transporters associated with MDR, making chemotherapeutic agents extracellular. To reduce the amount of drug in cancer cells, and to make cancer cells acquire resistance to chemotherapeutic agents (Non-Patent Document 3).

On the other hand, many attempts have been made to develop a sensitizer that inhibits P-glycoprotein-mediated drug outflow and resensitizes cancer cells to a chemotherapeutic agent (Non-Patent Document 4). These sensitizers include calcium channel blockers, immunosuppressants, calmodulin antagonists (Non-Patent Document 5), and piperine (Non-Patent Document 6), which is the main component of black pepper. Unfortunately, most of these sensitizers have failed in clinical trials. It has been found that first-generation inhibitors are toxic to P-glycoprotein, non-specific, and interact with other cell transporters (Non-Patent Document 7). Although the second-generation inhibitors were less toxic, they interacted with cellular enzymes and affected the metabolism of chemotherapeutic agents (Non-Patent Document 8).
Therefore, there is a need for a sensitizer that is non-toxic, does not affect the metabolism of chemotherapeutic agents, and suppresses multidrug resistance (MDR) due to P-glycoprotein.

In recent years, we have described nanoparticles composed of a complex of _{polyethylene glycol (PEG) and TiO 2 (hereinafter sometimes referred to as TiO 2-} PEG nanoparticles) of hepatocyte growth factor receptor (HGFR). It has been shown to promote the proliferation of HepG2 cell lines through aggregation (Non-Patent Documents 9 to 12). However, this report does not describe any multidrug resistance (MDR) due to P-glycoprotein.

JP-A-2019-18007 JP-A-2017-122684 JP 2010-180192 JP 2010-59143 WO2010 / 016581 JP-A-2009-91345 JP-A-2009-73784 Japanese Patent Application Laid-Open No. 2008-162995 Special table 2019-523757 Special table 2013-523680 WO2014 / 119568 Special table 2016-53618 WO2011 / 040503 JP-A-2009-247309 Special table 2016-21913 JP 2013-172731

Holohan C; Van Schaey broeck S, Longley DB and Johnston PG. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer. 2013, 13, 714-726. Ward A, Reyes CL, Yu J, Roth CB and Chang G. Flexibility in the ABC transporter MsbA: Alternating access with a twist. Proc. Natl. Acad. Sci. U.S. A. 2007, 104, 19005-19010. Gillet JP and Gottesman MM. Mechanisms of multidrug resistance in cancer. Methods. Mol. Biol. 2010, 596, 47-76. Harmsen, S, Meijerman I, Febus CL, Maas-Bakker RF, Beijnen JH and Schellens JH. PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line. Cancer Chemother. Pharmacol. 2010, 66 , 765-771. Harmsen, S, Meijerman I, Febus CL, Maas-Bakker RF, Beijnen JH and Schellens JH. PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line. Cancer Chemother. Pharmacol. 2010, 66 , 765-771. Chinta G, Safiulla BS, Mohane SC and Latha P. Piperine: A Comprehensive Review of Pre-Clinical and Clinical Investigations. Curr. Bioact. Comp.2015, 11, 156-169. [19] Safiulla Basha Syed et al., “ Targeting P-glycoprotein: Investigation of piperine analogs for overcoming drug resistance in cancer ”, Scientific REPORTS 7:7972, published online: 11 August 2017 Krishna R and Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci. 2000, 11, 265-283. Amin ML. P-glycoprotein Inhibition for Optimal Drug Delivery. Drug Target Insights. 2013, 7, 27-34. Qingqing Sun, Koki Kanehira and Akiyoshi Taniguchi. “Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells”, Science and Technology of Advanced Materials. 2016, 17: 1, 669-676. Le Thi Minh Phuc and Akiyoshi Taniguchi. Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis. Int. J. Mol. Sci. 2017, 18, 1301. Alaa Fehaid and Akiyoshi Taniguchi. Size-dependent effect of silver nanoparticles on the tumor necrosis factor α-induced DNA damage response Int. J. Mol. Sci. 2019, 20, 1038. Qingqing Sun, Koki Kanehira, Akiyoshi Taniguchi. Uniform TiO2 nanoparticles induce apoptosis in epithelial cell lines in a size-dependent manner, Biomaterials Science. 2017, 5, 1014-1021. Kazuhiro Katayama et al., “Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway”, Biochimica et Biophysica Acta, Volume 1853, October 2015, Pages 2361-2370 Cheol-Hee Choi, “ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal”, Cancer Cell International 2005, 5:30

The present invention improves the problems of conventional P-glycoprotein inhibitors described above, more specifically, for anti-cancer agents of cancer or tumor cells without toxicity and without affecting the metabolism of chemotherapeutic agents. It is an object of the present invention to provide a pharmaceutical composition that prevents or reduces resistance, particularly multidrug resistance (MDR).

When the present inventors administer an anticancer drug to tumor cells expressing P-glycoprotein in combination with nanoparticles having a predetermined particle size composed of a material that is not cytotoxic and does not affect cell metabolism, We have found that the excretion of an anticancer drug from cancer or tumor cells is reduced, the anticancer drug is retained in the cells, and the anticancer effect can be enhanced, leading to the present invention.
The present invention is based on such findings, and provides the following pharmaceutical compositions and the like.

[1] A pharmaceutical composition for preventing or reducing resistance to a chemotherapeutic agent, which comprises nanoparticles made of a biocompatible material having an average particle size of 300 nm or less.
[2] The pharmaceutical composition according to [1], which is used for treating cancer or tumor in which P-glycoprotein is expressed on the surface of cancer or tumor cells.
[3] The pharmaceutical composition according to [1] or [2], wherein the nanoparticles have an average particle size of 10 to 200 nm.
[4] The pharmaceutical composition according to any one of [1] to [3], which is used in combination with the chemotherapeutic agent.
[5] The pharmaceutical composition according to any one of [1] to [3], which comprises the chemotherapeutic agent.
[6] The pharmaceutical composition according to any one of [1] to [5], which is administered within 24 hours before and after administration of the chemotherapeutic agent.
[7] The cancer or tumor includes liver cancer, testicle tumor, kidney cancer, bladder cancer, renal pelvis / urinary tract tumor, urinary tract epithelial cancer, prostate cancer, breast cancer, ovarian cancer, head tumor, cervical tumor, head and neck. Cancer, brain tumor, lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant pleural mesoderma, mesoderma), colon cancer, esophageal cancer, colon cancer, colorectal cancer, gastric cancer, anal cancer, osteosarcoma, embryo Cellular tumors (testis tumors, ovarian tumors, extragonadal tumors), cervical cancer, uterine body cancer, central nervous system cancer, retinal cancer, hematological cancer, neuroblastoma, multiple myeloma, malignant bone tumor, soft tissue sarcoma , Skin cancer, connective tissue cancer, fatty cancer, biliary tract cancer, acute lymphoma, lymphoid cancer, relapsed / refractory malignant lymphoma, pancreatic cancer, and pediatric malignant solid tumor (horizontal myoma, neuroblastoma, hepatoblastoma, etc.) The pharmaceutical composition according to any one of [1] to [6], which is selected from the group consisting of primary malignant tumors of the liver, myeloma, etc.).
[8] The pharmaceutical composition according to [7], wherein the cancer is selected from lung cancer, pancreatic cancer, ovarian cancer, breast cancer and colon cancer.
[9] The pharmaceutical composition according to any one of [1] to [8], wherein the cancer or tumor is resistant to the chemotherapeutic agent.
[10] The pharmaceutical composition according to any one of [1] to [9], wherein the cancer is a recurrent or metastatic cancer.
[11] The nanoparticles are at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene, or a biocompatible polymer thereof. The pharmaceutical composition according to any one of [1] to [10], which comprises the complex of.
[12] The complex has the biocompatibility on the surface of particles consisting of at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene. The pharmaceutical composition according to [11], to which a sex polymer is bound.
[13] The biocompatible polymer according to [11] or [12], wherein the biocompatible polymer is at least one water-soluble polymer selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyethylene oxide, and dextran. Pharmaceutical composition.
[14] The pharmaceutical composition according to [12] or [13], wherein the complex is a complex in which the biocompatible polymer is bound to the surface of particles made of titanium oxide.
[15] The pharmaceutical composition according to any one of [1] to [14], wherein the chemotherapeutic agent is a DNA synthesis inhibitor, a cell division inhibitor, or a DNA damaging agent.
[16] The chemotherapeutic agent includes etoposide, cisplatin, oxaliplatin, gemcitabine, methotrexate, leucovorin, pemetrexed disodium, camptothecin, doxorubicin, vinblastine, vincristine, bindesin, citarabin, azathiopurine, melphalan, imatinib, anastrozol. From TOPK inhibitors such as sol, carboplatin, paclitaxel, docetaxel, vincristine, mitoxantrone, artesnate, capecitabin, etoposide, binorerbin, OTS964, NLG919, OTS167 and OTSC41, as well as their pharmacologically acceptable salts or prodrugs. The pharmaceutical composition according to any one of [1] to [15], which comprises at least one selected from the group.
[17] The chemotherapeutic agent is used in combination with a cancer treatment selected from the group consisting of surgery, photodynamic therapy, radiotherapy, immunotherapeutic agent, cryotherapy and nucleic acid chemotherapeutic agent [1] to [16]. ] The pharmaceutical composition according to any one of.

Here, some terms used in the present specification are also defined.
As used herein, "biocompatibility" or "biocompatibility" means that it is not cytotoxic and does not affect cell metabolism.
In the present specification, the "average particle size" means the average value of the particle size analyzed by a particle size distribution meter of a dynamic light scattering method (for example, nanoSAQLA, manufactured by Otsuka Electronics Co., Ltd.).
In the specification of the present application, "p-glycoprotein is expressed on the cell surface" is analyzed by flow cytometry, immunostaining, etc., and the p-glycoprotein present on the tumor cell is compared with the corresponding normal cell. Therefore, it means that the amount has increased by 1.5 times or more.

Figure 1 shows the effect of HepG2 cell line and effect of _TiO 2-PEG nanoparticles on A431 cell lines, as well as HepG2 cell lines and _TiO 2-PEG nanoparticles into cisplatin cytotoxicity to A431 cell line. Figure 1A in the absence of cisplatin, show the viability of the HepG2 cell line when exposed HepG2 cells lines to _TiO 2-PEG nanoparticles. Figure 1C, in the presence of cisplatin, show the viability of the HepG2 cell line when exposed HepG2 cells lines to _TiO 2-PEG nanoparticles. Figure 1B is the absence of cisplatin, show the viability of the A431 cell lines when exposed to A431 cell lines _TiO 2-PEG nanoparticles. Figure 1D in the presence of cisplatin, show the viability of the A431 cell lines when exposed to A431 cell lines _TiO 2-PEG nanoparticles. Black circles is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle size of 100 nm, white circles is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle diameter of 200 nm, black squares, it is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle size of 300 nm. All values were normalized to control untreated cells. All values are shown as mean ± SD (n ≧ 3). Data were analyzed using Student's t-test. FIG. 2 _{shows the effect of TiO 2-} PEG nanoparticles on cisplatin uptake by the HepG2 and A431 cell lines. 2A shows the concentration of cisplatin incorporated in HepG2 cell line, white _bars indicate the concentration of intracellular cisplatin in the absence of TiO 2-PEG nanoparticles, black _bars, TiO 2-PEG nanoparticles It shows the concentration of intracellular cisplatin in the presence of particles. The calibration curve prepared by using a dilution series of platinum standard solution, the correlation coefficient (R ²⁾ was 1.0. All values are shown as mean ± SD (n ≧ 3). Data were analyzed using Student's t-test. ** p ≤ 0.01. 3, on the uptake of _TiO 2-PEG nanoparticles by HepG2 cell line and A431 cell _lines, showing the effect of the size and concentration of the TiO 2-PEG nanoparticles. FIG. 3A shows the test results on the HepG2 cell line, and FIG. 3B shows the test results on the A431 cell line. Black circles is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle size of 100 nm, white circles is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle diameter of 200 nm, black squares, it is a graph of the case of adding _TiO 2-PEG nanoparticles with an average particle size of 300 nm. The vertical axis shows the percentage of cells incorporating nanoparticles. All values are shown as mean ± SD (n ≧ 3). Data were analyzed using Student's t-test. ** p ≤ 0.01. FIG. 4 _{shows the effect of TiO 2-} PEG nanoparticles on the localization and expression of P-glycoprotein in the HepG2 and A431 cell lines. Figure 4A, when treated with cisplatin IC50 values HepG2 cell lines in the absence of _TiO 2-PEG nanoparticles with _(top), when treated with cisplatin IC50 values in the presence of TiO 2-PEG nanoparticles (Lower) shows a nuclear-stained image with DAPI (left), an immunofluorescent-stained image with an anti-P-glycoprotein antibody (middle), and an integrated image (right). Figure 4B, when treated with cisplatin IC50 value A431 cell line in the absence of _TiO 2-PEG nanoparticles with _(top), when treated with cisplatin IC50 values in the presence of TiO 2-PEG nanoparticles (Lower) shows a nuclear-stained image with DAPI (left), an immunofluorescent-stained image with an anti-P-glycoprotein antibody (middle), and an integrated image (right).

The pharmaceutical composition of the present invention prevents or reduces resistance to a chemotherapeutic agent to enhance the anticancer activity of the chemotherapeutic agent, and provides nanoparticles made of a biocompatible material having a predetermined average particle size. It is characterized by including.

The nanoparticles contained in the pharmaceutical composition of the present invention have an average particle size of 300 nm or less. As shown in Examples described later, when small particles of this level coexist with an anticancer drug, the excretion of the anticancer drug by cancer or tumor cells to the outside of the cell is suppressed, the anticancer drug is retained in the cells, and the anticancer drug is retained. Cancer action is enhanced.
As the nanoparticles of the present invention, nanoparticles having an average particle size of 10 to 200 nm are preferable, and nanoparticles having an average particle size of 10 to 100 nm are particularly preferable, in that the anticancer effect is further enhanced.

The nanoparticles of the present invention are not cytotoxic by themselves, preferably do not affect cell metabolism, and are preferably composed of biocompatible materials.
Therefore, in a preferred embodiment, the nanoparticles are at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene, or biocompatibility with them. It comprises a complex with a polymer and is preferably composed substantially of these materials. Among these, at least one selected from the group consisting of titanium oxide, silver, gold, and polystyrene nanoparticles, or a composite of these and a biocompatible polymer is preferable.

The biocompatible polymer is not particularly limited and may be an anionic, cationic or nonionic biocompatible polymer, for example, a carboxyl group, an amino group, a diol group, a salicylate group, a phosphoric acid group, etc. Examples thereof include biocompatible polymers having at least one functional group selected from a hydroxyl group and a polyoxyalkylene group.

Anionic biocompatible polymers are typically biocompatible polymers with carboxyl groups, such as carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polycarboxylic acids, and copolymers with carboxyl group units. Examples include polymers (copolymers). From the viewpoint of hydrolyzability and solubility in the living body, polycarboxylic acids such as polyacrylic acid and polymaleic acid, and copolymers (copolymers) of acrylic acid / maleic acid and acrylic acid / sulfonic acid-based monomers are preferable. Acrylic acid is more preferred.

From the viewpoint of dispersibility, the anionic biocompatible polymer is preferably a polymer having a weight average molecular weight of 2000 to 100,000, more preferably a polymer having a weight average molecular weight of 5,000 to 40,000, and further preferably a polymer having a weight average molecular weight of 5,000 to 20,000. The weight average molecular weight of the biocompatible polymer is a value measured by size exclusion chromatography (hereinafter, the same applies).

The cationic biocompatible polymer is typically a biocompatible polymer having an amino group, and examples thereof include polyamino acids, polyamines, and copolymers having amine units. From the viewpoint of hydrolyzability and solubility in a living body, polyamines such as polyethyleneimine, polyvinylamine and polyallylamine are preferable, and polyethyleneimine is more preferable.

From the viewpoint of dispersibility, the cationic biocompatible polymer is preferably a polymer having a weight average molecular weight of 2000 to 100,000, more preferably a polymer of 5,000 to 40,000, and further preferably a polymer of 5,000 to 20,000.

The nonionic biocompatible polymer preferably includes a polymer having a hydroxyl group and / or a polyoxyalkylene group. Examples of such biocompatible polymers include polyoxyalkylene glycols, dextran, and copolymers containing them, with polyoxyalkylene glycols or dextran being more preferred, and polyoxyalkylene glycol being even more preferred.
Examples of polyoxyalkylene glycol include polytetramethylene ether glycol, polyethylene glycol, polyethylene oxide, polypropylene glycol, polyoxyethylene polyoxypropylene glycol, and polyvinyl alcohol. Among them, polyethylene oxide, polyvinyl alcohol, and polyethylene glycol are used. Preferably, polyethylene glycol is more preferred.

From the viewpoint of dispersibility, the nonionic biocompatible polymer is preferably a polymer having a weight average molecular weight of 2000 to 100,000, and more preferably a polymer having a weight average molecular weight of 5,000 to 40,000.

In a preferred embodiment, the nanoparticles of the present invention have biocompatibility with at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene. It comprises a complex with a polymer and is preferably composed substantially of the complex.
The composite is typically formed on the surface of particles of at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene. Biocompatible polymers are bound. The particles can bind components other than the biocompatible polymer, but in the present invention, the nanoparticles themselves are preferably not cytotoxic and have properties that do not affect cell metabolism. It is preferable that the other components have the same characteristics, and it is more preferable that the other components are not contained.

In the nanoparticles of the present invention, the mode in which the biocompatible polymer is bonded to the particle surface is not particularly limited, and may be a chemical bond such as a covalent bond or a coordination bond, or a hydrophobic bond or the like. It may be a physical bond such as adsorption using affinity or structural involvement such as entanglement. Typically, at least one selected from the functional groups of the biocompatible polymers described above, preferably carboxyl groups, amino groups, diol groups, salicylic acid groups, phosphate groups, hydroxyl groups and polyoxyalkylene groups. Nanoparticles in which a biocompatible polymer is bonded to the surface of the particles via a functional group are mentioned as a preferred embodiment.

The state in which the biocompatible polymer is bonded to the particle surface is a state in which the biocompatible polymer is bonded to a part of the particle surface, and most or the entire surface of the particle is covered with the biocompatible polymer. Includes the state of being broken. As an example of the former, a biocompatible polymer on the surface of particles via at least one functional group selected from a carboxyl group, an amino group, a diol group, a salicylic acid group, a phosphoric acid group, a hydroxyl group and a polyoxyalkylene group. In the latter case, an inorganic particle such as alumina is chemically bonded to an organic resin such as a vinyl group, an epoxy group, an amino group, a (meth) krill group, an isocyanate group or a mercapto group. Examples thereof include a complex obtained by reacting a silane coupling agent having a functional group that can be formed, and then reacting with a biocompatible polymer.

The titanium oxide may be any of anatase type titanium oxide, rutile type titanium oxide and amorphous type titanium oxide, and amorphous type titanium oxide is preferable.
The polystyrene is not particularly limited, but polystyrene having a weight average molecular weight of 10 ³ to 10 ⁷ is usually used.

The anticancer agent used in combination with the nanoparticles of the present invention is a chemotherapeutic agent in which resistance to cancer or tumor cells can be a problem, and includes most chemotherapeutic agents. Many chemotherapeutic agents inhibit cell division of cancer or tumor cells by inhibiting DNA synthesis, inhibiting cell division, or DNA damage to treat the cancer or tumor or slow the progression of symptoms. These chemotherapeutic agents are taken up into cells and act on the cell nucleus to exert their effects, but cancer or tumor cells excrete anticancer agents to the outside of the cells by P-glycoprotein and acquire resistance. The nanoparticles of the present invention can reduce the expression of such P-glycoprotein on the cell surface, or promote the translocation of P-glycoprotein from the cell surface into the cell and inhibit the excretion of anticancer agents. it can. Therefore, nanoparticles are applicable as long as they are anticancer agents that are excreted outside tumor cells by P-glycoprotein and resistance can be a problem.

Therefore, the anti-cancer agents used in combination with the nanoparticles of the present invention include most chemotherapeutic agents, such as etoposide, cisplatin, oxaliplatin, gemcitabine, methotrexate, leucovorin, pemetrexed disodium, camptothecin, doxorubicin, vinblastine, vincristine, TOP , And at least one selected from the group consisting of these pharmacologically acceptable salts or prodrugs.
Among them, the nanoparticles of the present invention are effective for cisplatin, paclitaxel and docetaxel, in which the acquisition of drug resistance can be a particular problem.

In some embodiments, the anti-cancer agent is a polymer micelle, liposome, dendrimer, polymer-based nanoparticles, silica-based nanoparticles, nanoscale-coordinating polymer, or a formulation in which the active ingredient is bound or encapsulated in inorganic nanoparticles. It may be in the form of.

The pharmaceutical composition of the present invention contains the nanoparticles and is used in combination with the anticancer agent. Therefore, the pharmaceutical composition of the present invention may contain the nanoparticles and the anticancer agent, but may be prepared separately from the anticancer agent. In this case, the pharmaceutical composition of the present invention may be administered at the same time as the anticancer drug, but may be administered before or after the administration of the anticancer drug. In order to obtain the effect of enhancing the anticancer effect of the nanoparticles of the present invention, it is preferable that the anticancer agent and the nanoparticles of the present invention are administered so as to coexist around the cancer or tumor tissue. It is preferable to administer the pharmaceutical composition of the present invention within 24 hours before and after administration, and more preferably at the same time. However, the timing of administration can be appropriately adjusted according to the patient's condition, the administration method of the nanoparticles and the anticancer agent, the type of cancer or tumor, etc., in consideration of the half-life of the nanoparticles and the anticancer agent to be used.

In some embodiments, the compositions of the invention may comprise a pharmaceutically acceptable carrier and other components.
For example, antioxidants, buffers, bacteriostatic agents, bactericidal antibiotics, suspending agents, thickeners and the like can be included. However, without being limited to these, the compositions of the present invention may contain other components customarily used in the art.

The anti-cancer agent used in combination with the nanoparticles of the present invention can be further combined with other therapeutic means for cancer. Examples of such other therapeutic means for cancer include photodynamic therapy, immunotherapy, nucleic acid chemotherapy, surgical treatment, radiotherapy and the like.

In some embodiments, the immunotherapeutic agent includes anti-CD52 antibody, anti-CD20 antibody, anti-CD47 antibody, anti-GD2 antibody, cytokine and the like, and specific examples thereof include alemtuzumab, ofatumumab, rituximab, zevalin and ADCETRIS. , Cadecil, Ontaku, etc., but are not limited to these. Examples of cytokines include, but are not limited to, interferon and interleukin, and more specifically, IFN-α, IFN-γ, IL-2, IL-12, and TNF-α.
From the viewpoint of action, PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, IDO inhibitor, CCR7 inhibitor, OX40 inhibitor, TIM3 inhibitor, LAG3 inhibitor inhibitor, etc. However, it is not limited to these.
In addition, the immunotherapeutic agent includes an antibody bound to a cell-killing radiation source and an antibody bound to an active substance having anticancer activity.

Nucleic acid chemotherapeutic agents include siRNA, miRNA and antisense oligonucleotides.
The siRNA includes siRNA that targets a gene encoding an anti-apoptotic protein, siRNA that targets a multidrug resistance (MDR) gene (eg, ABCB1, ABCB4, and ABCB5), and a gene that encodes a protein related to DNA repair. Examples include siRNAs that target siRNAs and siRNAs that target other pathways.
Specifically, for example, EGFR / ErbB1 siRNA, ErbB2 / HER2 / Neu siRNA, IGF-1R siRNA, K-ras siRNA, R-ras siRNA, BRAF siRNA, ABL siRNA, c-Src siRNA, Met siRNA, c- Myc siRNA, N-Myc siRNA, Cyclone-D1 siRNA, PI3K siRNA, AKT siRNA, NF-κβ siRNA, EWS / FLI-1 siRNA, HIF siRNA, HPV E7 siRNA, E2F4 siRNA, HPV E6 siRNA, Hdmx siRNA, Notch-1 siRNA, delta-like 1 siRNA, FLIP siRNA, BCL-2 siRNA, BCL-XL siRNA, surviving siRNA, XIAP siRNA, telomerase siRNA, ID1 siRNA, Cks-1 siRNA, Skp-2 siRNA, catepsin L siRNA, VEGF siRNA, EGF siRNA, FGF siRNA, PDGF siRNA, IL-8 siRNA, IGF-1 siRNA, catepsin siRNA, MMP2 siRNA, stromelysin siRNA, uPA siRNA, c-myc siRNA, ras siRNA, c-src siRNA, v-raf siRNA, c-jun siRNA, VEGFR siRNA, thymidine siRNA, phosphorylase siRNA, RAS-farnesyl siRNA, transferase siRNA, geranyl siRNA, transferase siRNA, IL-1 siRNA, IL-6 siRNA, IL-8 siRNA, Ang-1 siRNA, angiostatin Includes, but is not limited to, II siRNA, endoserin siRNA, iNOS siRNA, PAF siRNA, Cox-2 siRNA, ABCB1 siRNA, ABCB4 siRNA, ABCB5 siRNA, P-sugar protein siRNA, ERCC1 siRNA, and ATM siRNA. Of these, siRNAs that target multidrug resistance (MDR) genes (eg, ABCB1, ABCB4, and ABCB5) are preferred.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that control the translation and stability of mRNA. MicroRNAs (miRNAs) associated with cancer or tumor treatment include iR-9, miR-15, miR-16, miR-34, miR-181, miR-200, miR 200c, miR-342, miR-630. , Let-7, LIN28, DICER, miR-155, miR-21, and miR-17-29, but are not limited to these. For example, it is known that forced expression of miR-9 suppresses SOX2 expression and results in a decrease in ABC transporter expression.

Antisense oligonucleotides are unmodified or chemically modified single-stranded DNA molecules that generally hybridize to mRNAs that are relatively short (13-25 nucleotides) and have unique sequences that are present in the cell. It inhibits the translation of mRNA. Gene targets for antisense oligonucleotides associated with the treatment of cancer or tumors include Bcl-2, Survivin, MDM2, Bcl-XL, RelA, RAS, RAF, BCR-ABL, JNK1,2, TERT, c-myc, And c-myb, but not limited to these,

The pharmaceutical composition of the present invention can be widely used for cancers or tumors to which a chemotherapeutic agent is applied, and particularly for cancers or tumors in which P-glycoprotein is excessively expressed on the surface of cancers or tumor cells. Be done.
Examples of cancers or tumors to which the pharmaceutical composition of the present invention can be used include liver cancer, testicle tumor, kidney cancer, bladder cancer, renal pelvis / urinary tract tumor, urinary tract epithelial cancer, prostate cancer, breast cancer, ovarian cancer, head. Tumors, cervical tumors, head and neck cancers, brain tumors, lung cancers (eg, non-small cell lung cancer, small cell lung cancer, malignant pleural mesenteric tumor, mesenteric tumor), colon cancer, esophageal cancer, colon cancer, colonic rectal cancer, gastric cancer , Anal cancer, osteosarcoma, embryonic cell tumor (testis tumor, ovarian tumor, extragonadal tumor), cervical cancer, uterine body cancer, central nervous system cancer, retinal cancer, hematological cancer, neuroblastoma, multiple myeloma , Malignant bone tumor, soft tissue sarcoma, skin cancer, connective tissue cancer, fatty cancer, biliary tract cancer, acute lymphoma, lymphoid cancer, relapsed / refractory malignant lymphoma, pancreatic cancer, and pediatric malignant solid tumor (horizontal myoma, Neuroblastoma, hepatic blastoma and other primary malignant tumors of the liver, medullary blastoma, etc.). Among them, it is preferably used for liver cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and colon cancer in which multidrug resistance is particularly problematic.

Also, from the same viewpoint, in particular, when treating a cancer or tumor that has already acquired drug resistance, a recurrent cancer, or a metastatic cancer with a chemotherapeutic agent, the pharmaceutical composition of the present invention is combined. Therefore, it is expected that the anti-cancer effect of the chemotherapeutic agent will be enhanced.

The pharmaceutical compositions of the present invention can be used in vitro, for example, in contact with isolated cells or tissues, or administered in vivo to a living organism such as a patient. The subject to which the pharmaceutical composition of the present invention is administered may include all vertebrate species, but is often a mammal, most commonly used in human patients.
In addition, the patient is a person suffering from cancer or tumor, but preferably a person suffering from cancer or tumor in which P-glycoprotein can or is highly expressed on the surface of cancer or tumor cells. Is.
Patients can also be those who have not yet received chemotherapy and those who have already received chemotherapy. For example, the composition of the present invention can be administered to a patient after surgery or radiation therapy in combination with a chemotherapeutic agent. In addition, the composition of the present invention may be administered in combination with a chemotherapeutic agent as maintenance therapy for a patient who has already received chemotherapy and a therapeutic effect has been observed. In addition, the composition of the present invention is administered in combination with a chemotherapeutic agent to a patient who has already received chemotherapy but whose cancer has recurred or metastasized, and / or a patient who has been found to be resistant to the chemotherapeutic agent. May be good.

The pharmaceutical composition of the present invention is not limited in terms of administration method, for example, intravenous administration, intratumoral injection, oral administration, subcutaneous administration, intraperitoneal injection, intracranial injection, rectal administration, and spraying into the pulmonary pathway. The specific mode of administration depends on various factors including the distribution and abundance of the cells to be treated, and the mechanism of metabolism or removal of the composition in the migration route from the administration site. Is decided. For example, relatively superficial tumors are preferably selected for intratumoral injection, and internal tumors are preferably given intravenous injection. Further, the method of administering the pharmaceutical composition of the present invention may be the same as or different from the method of administering the chemotherapeutic agent which is also referred to. This is also determined depending on the factors mentioned above.

The dose of the pharmaceutical composition of the present invention may be an amount capable of enhancing the anticancer effect of the combined chemotherapeutic agent, and may be an amount capable of obtaining a desired effect depending on the type of cancer and the type of chemotherapeutic agent. .. However, administration at a low dose is assumed from the test results in the examples described later. Specifically, in the examples, when cisplatin was used in combination with cisplatin at a CH50 value of cisplatin against a HepG2 cell line, that is, 12 μg / ml, the most remarkable anticancer effect was obtained with 10 μg / ml nanoparticles, and this test was conducted. The dose in the clinical trial is determined by referring to the result and the information such as the dose in the package insert of the chemotherapeutic agent whose clinical dose of cisplatin etc. is already known, and the desired dose is determined from the result of the clinical trial. It is expected that the effective dose will be determined. However, the actual dose of the pharmaceutical composition of the present invention will be determined by those skilled in the art due to various factors such as the patient's condition, cancer type, administration method, dosage form, other therapeutic methods combined with chemotherapy, and the condition of cancer tissue. Can be adapted in consideration of.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

1. Preparation of nanoparticles Please add. Titania particles (Wako Pure Chemical Industries, Ltd.) were prepared into nanoparticles by a pressure pulverization method. The 25% suspension was pressurized at 200 MPa 10 times. Then, it was reacted with polyethylene glycol-maleic acid to prepare nanoparticles. The average particle size of the nanoparticles was analyzed using a dynamic light scattering particle size distribution meter (nanoSAQLA) manufactured by Otsuka Electronics Co., Ltd.

2. Evaluation test 2-1. Materials and Methods 2-1-1. Preparation of cisplatin.
Cisplatin [cis di ammine di chloro platinum (ii)] was obtained from FUJIFILM (Wako, Co., Ltd., Japan). A 0.5 mg / ml stock solution was prepared by dissolving cisplatin powder in 0.9 M NaCl solution ^{, stored at 4 o} C and used within 1 week. At the time of use, the stock solution was diluted with culture medium to obtain the desired final concentration.

2-1-2. Cell Lines and Cell Culture Two different cell lines were used: HepG2 cell line derived from hepatocellular carcinoma and A431 cell line derived from epithelial cell carcinoma. High glucose Dalveco modified Eagle supplemented with 10% (v / v) heated fetal bovine serum (Biowest, USA), 100 μg / ml penicillin, and 10 μg / ml streptomycin (Nacalai Tesque, Japan) in HepG2 and A431 cell lines. The cells were cultured in medium (DMEM, high glucose, Nacalai Tesque, Japan) at 37 ° C. and 5% CO ₂ . Cells were subcultured every 2 days to reach a 70-80% confluent state.

2-1-3. Statistical analysis All data from the following studies were evaluated for statistical significance using the Student's t-test. All values are expressed as mean ± standard deviation using 3 or more independent iterations (n ≧ 3). * P ≤ 0.05, ** p ≤ 0.01.

2-2. Determination of Cisplatin 50% Inhibition Concentration (IC50) Values Prior to testing, cisplatin IC50 values for HepG2 and A431 cell lines were measured with the LIVE / DEAD® Viability / Cytotoxicity Kit (Invitrogen, Ltd,). UK) was used and evaluated according to the manufacturer's instructions according to the fluorescent microplate protocol. Briefly, 1 × 10 ⁴ cells / well were seeded on Costar 96 well plates and incubated at _{37 ° C. at 5% CO 2.} After 24 hours, cells were exposed to different concentrations of cisplatin for 24 hours. The cells were then stained with 1 μM calcein AM for viable cells and 2 μM ethidium homodimer 1 (EthD-1) for dead cells, and the fluorescence intensity was determined by Spark ™ 10M multimode microplate reader (Tecan Ltd.,). Measured by Switzerland). IC50 values were determined from the dose-response curve. The average IC50 value of cisplatin for each type of cell line was calculated from three independent experiments, each performed three times.
The average IC50 value of cisplatin was 12 μg / ml for the HepG2 cell line and 6 μg / ml for the A431 cell line.

2-3. The effect of _TiO 2-PEG nanoparticles on viability and cisplatin cytotoxicity effects HepG2 cell line and A431 cell lines _TiO 2-PEG nanoparticles cell viability and to cisplatin cytotoxicity, by fluorescence microplate protocol Evaluation was performed using the LIVE / DEAD® Viability / Cytotoxicity Kit (Invitrogen, Ltd, UK) according to the manufacturer's instructions. Briefly, 1 × 10 ⁴ cells / well were seeded in Costar 96 wells and incubated at _{37 ° C. and 5% CO 2 conditions.} After 24 h, cells, 100 nm of different concentrations (0,10,40,100 and 400 [mu] g / ml medium), the _TiO 2-PEG nanoparticles 200nm or 300 nm, in the presence or absence of cisplatin IC50 values , 24 hours exposure. The cells were then stained with 1 μM calcein AM for viable cells and with 2 μM ethidium homodimer 1 (EthD-1) for dead cells and then fluorescent with a Spark ™ 10 M multimode microplate reader (Tecan Ltd., Switzerland). The intensity was measured.

As shown in FIG. 1A, 3 different sizes of the _TiO 2-PEG nanoparticles increased the survival rate of the comparison to HepG2 cell lines with control untreated cells. Nanoparticles with an average particle size of 10 μg / ml increased cell viability up to 160%, showing the most remarkable effect. The 300 nm nanoparticles increased cell viability up to 120%. These results were the same as the results previously reported by the present inventors (Non-Patent Document 10).
As shown in FIG. 1C, a similar tendency was observed for the A431 cell line.
These results indicate that the TiO _2- PEG nanoparticles induce cell proliferation in any tumor cell, and that the smaller the particle size, the greater the cell proliferation effect, especially at low concentrations. _Further, TiO 2-PEG nanoparticles themselves show that for both cells without cytotoxic effects.
_{Regarding the effect of TiO 2-} PEG nanoparticles on cisplatin cytotoxicity, as shown in FIGS. 1B and 1D, in the _{absence of TiO 2-} PEG nanoparticles in any of the cells, the cisplatin-treated cells were the same as the control untreated cells. By comparison, the survival rate was reduced to about 50%.
The HepG2 cell line, as shown in FIG. 1B, in the case of the TiO _2-PEG nanoparticles were treated in combination with cisplatin IC50 value, as compared with the case of treatment with cisplatin alone, cell viability was significantly reduced .. In addition, when 10 μg / ml nanoparticles of 100 nm were combined, the cell viability was reduced to 20%, showing the most remarkable effect.
The A431 cell line, as shown in FIG. _1D, cells treated with a combination of TiO 2-PEG nanoparticles and cisplatin, compared to control untreated cells showed an increase in _survival, TiO 2-PEG nanoparticles It was suggested that the particles did not affect the cisplatin cytotoxicity in the A431 cell line.
These results showed that TiO _2- PEG nanoparticles, especially 100 nm TiO _2- PEG nanoparticles, increased cisplatin cytotoxicity against the HepG2 cell line. On the other hand, TiO _2- PEG nanoparticles had no effect on cisplatin cytotoxicity against the A431 cell line.

2-4. Evaluation of Cellular Sisplatin Uptake by ICP-MS Analysis Cisplatin uptake by HepG2 and A431 cell lines was evaluated depending on the intracellular platinum concentration measured by inductively coupled plasma mass spectrometry (ICP-MS). Briefly, 10 ⁶ cells / well were seeded in 24-well plates and incubated at a 37 ° C. and 5% CO ₂ condition. After 24 hours, the cells, alone or after the _TiO 2-PEG nanoparticles 100nm were mixed in a concentration of 10 [mu] g / ml culture medium, and exposed to cisplatin IC50 values. After 24 hours, cells were washed with PBS, trypsinized, washed twice again PBS, trypan blue-stained, and counted the number of cells, were suspended 10 ⁶ cells in PBS for 1 mL, until digested Stored at 4 ° C. The sample was transferred to a quartz beaker, 1.5 ml HCL and 5 ml HNO ₃ were added, and then the sample was dried by heating. After cooling, 1.5 ml of HCl and 5 ml of HNO ₃ were further added, heated to completely decompose, and then the obtained solution was poured into a polypropylene container and diluted with Milli-Q water to 25 ml. A calibration curve was prepared using a standard solution prepared by serially diluting a standard solution (Kanto Chemical Co., Inc.), and the platinum concentration was measured by the Pb internal standard method by ICP-MS. Cell cisplatin uptake was expressed as [mu] g / 10 ⁶ cells.

As shown in FIG. _2A, HepG2 cell lines cisplatin treatment in the presence of TiO 2-PEG _{nanoparticles,} as compared to cisplatin treated cells in the absence of TiO 2-PEG nanoparticles, intracellular cisplatin level Has increased .
On the other hand, in the A431 cell line, as shown in FIG. _2B, TiO 2-PEG nanoparticles did not affect intracellular cisplatin level.

_{2-5. Evaluation of Cell Uptake of TiO 2-} PEG Nanoparticles by Flow Cytometry The _{uptake of dio 2-} PEG nanoparticles by HepG2 and A431 cell lines was evaluated depending on the change in light scattering by flow cytometry. Briefly, 10 ⁶ cells / well were seeded in 24-well plates, incubated for 24 hours at the 37 ° C. and 5% CO ₂ condition and then the cells with various concentrations (0,10,40,100 and 400μg (/ Ml medium) was exposed to _{TiO 2} PEG nanoparticles having an average particle size of 100 nm, 200 nm and 300 nm. After 24 hours, cells were washed twice, trypsinized, washed 3 times with PBS, dispersed in 1 mL of 6% HFBS / PBS aqueous solution, stored on ice and analyzed within 1 hour. Immediately prior to analysis, cells were passed through a nylon mesh (Cell Strainer Snap Cap, Falco, USA) and then the intracellular particle size was assessed using laterally scattered light by a P6800 spectrum analyzer (Sony Biotechnology, Japan) and anterior. Cell size was evaluated using scattered light. The percentage of cells with nanoparticles was calculated based on changes in gate area compared to control untreated cells.

As shown in Figure 3A, the TiO _2-PEG nanoparticles uptake increased as increasing the size and concentration of nanoparticles. As shown in FIG. 3B, a similar tendency was observed in the A431 cell line. These results, in any cell lines, the TiO _2-PEG nanoparticles uptake showed that increases depending on the size and concentration of nanoparticles.
The _TiO 2-PEG nanoparticles with an average particle size of 100nm of 10 [mu] g / ml, cellular uptake was very low. _This indicated that most of the TiO 2-PEG nanoparticles was on the extracellular or cell surface, TiO _2-PEG nanoparticles, influence on some surface proteins play an important role in cisplatin cytotoxicity Suggests the possibility of exerting. _The protein TiO 2-PEG nanoparticles affects, not the A431 cell line, it was inferred to be present mainly in the cell surface of the HepG2 cell line.

2-6. Effect of TiO _2- PEG nanoparticles on the localization or expression of P-glycoprotein P-glycoprotein is a surface protein involved in the outflow of cisplatin and other chemotherapeutic agents to the outside of the cell and is intracellular. It results in reduced drug accumulation and induction of MDR. Therefore, in order to determine TiO _2-PEG nanoparticles how to influence the P- glycoprotein was examined the localization or expression of P- glycoprotein. Briefly, HepG2 and A431 cell lines were placed in a 4-compartment cell view cell culture dish (Greiner Bio-One, Inc., USA) at 37 ° C. and 5% CO ₂ at a concentration of 4 × 10 ⁴ cells / compartment. Was sown for 24 hours. The cells were then treated with IC50 value cisplatin for an additional 24 hours with or without 10 μg / ml medium of _{100 nm TiO 2-PEG nanoparticles, after which the cells were washed with PBS and 4% paraformaldehyde (PFA).} ) For 10 minutes, then permeable with 1% Triton X-100. The cells were then blocked with a solution containing 1% bovine serum albumin (BSA), 10% normal goat serum and 0.3M glycine in 0.1% Tween-PBS for 1 hour at room temperature, followed by anti-P-glycoprotein. Incubated overnight at 4 ° C. with protein mouse antibody (1/500 dilution, Abcum, UK). The cells were then washed 3 times with PBS (10 minutes each) and incubated with goat anti-mouse IgG H & L (1/500 dilution, Alexa Fluor® 488, Abcam, UK) in the dark at room temperature for 1 hour. , Then washed 3 times (10 minutes each) with PBS. Nuclear DNA was labeled with DAPI (Thermo Fisher Scientific, USA). Images were taken using a confocal laser scanning microscope (LSM510 META, Carl Zeiss Inc., Germany).

As shown in FIG. 4A, confocal microscopic images showed that P-glycoprotein was highly expressed on the surface of HepG2 cells. If the HepG2 cell lines were treated in combination with cisplatin and TiO _2-PEG nanoparticles, P- glycoprotein localized intracellularly, expressed on the cell surface was less.
On the other hand, in the A431 cell line, as shown in FIG. 4B, P- expression of the glycoprotein is very _low, TiO 2-PEG nanoparticles had no effect on the expression and localization of P- glycoprotein. These results showed that changes in P-glycoprotein localization and expression in the HepG2 cell line affect cisplatin cytotoxicity. TiO _2- PEG nanoparticles cause the translocation of P-glycoprotein from the cell membrane to the cytoplasm in the HepG2 cell line, or a decrease in the expression of P-glycoprotein on the cell membrane, and this change is the function of P-glycoprotein. It is believed to have resulted in inhibition and intracellular cisplatin retention.

Claims (17)

A pharmaceutical composition for preventing or reducing the resistance of cancer or tumor cells to a chemotherapeutic agent, which comprises nanoparticles made of a biocompatible material having an average particle size of 300 nm or less. The pharmaceutical composition according to claim 1, which is used for treating cancer or tumor in which P-glycoprotein is expressed on the surface of cancer or tumor cells. The pharmaceutical composition according to claim 1 or 2, wherein the nanoparticles have an average particle size of 10 to 200 nm. The pharmaceutical composition according to any one of claims 1 to 3, which is used in combination with the chemotherapeutic agent. The pharmaceutical composition according to any one of claims 1 to 3, which comprises the chemotherapeutic agent. The pharmaceutical composition according to any one of claims 1 to 5, which is administered within 24 hours before and after administration of the chemotherapeutic agent. The cancers or tumors include liver cancer, testicle tumor, kidney cancer, bladder cancer, renal pelvis / urinary tract tumor, urinary tract epithelial cancer, prostate cancer, breast cancer, ovarian cancer, head tumor, cervical tumor, head and neck cancer, brain tumor. , Lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant pleural mesopharyngeal tumor, mesenteric tumor), colon cancer, esophageal cancer, colon cancer, colon rectal cancer, gastric cancer, anal cancer, osteosarcoma, germ cell tumor ( Testicular tumor, ovarian tumor, extragonadal tumor), cervical cancer, uterine body cancer, central nervous system cancer, retinal cancer, hematological cancer, neuroblastoma, multiple myeloma, malignant bone tumor, soft tissue sarcoma, skin cancer , Bonded tissue cancer, adipose cancer, biliary tract cancer, acute lymphoma, lymphoid cancer, relapsed / refractory malignant lymphoma, pancreatic cancer, and pediatric malignant solid tumor (horizontal myoma, neuroblastoma, hepatoblastoma and other primary malignant liver) The pharmaceutical composition according to any one of claims 1 to 6, which is selected from the group consisting of tumors, myelomas, etc.). The pharmaceutical composition according to claim 7, wherein the cancer is selected from lung cancer, pancreatic cancer, ovarian cancer, breast cancer and colon cancer. The pharmaceutical composition according to any one of claims 1 to 8, wherein the cancer or tumor is resistant to the chemotherapeutic agent. The pharmaceutical composition according to any one of claims 1 to 9, wherein the cancer is a recurrent or metastatic cancer. The nanoparticles are at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene, or a composite of these with a biocompatible polymer. The pharmaceutical composition according to any one of claims 1 to 10, which comprises. The complex is formed on the surface of particles consisting of at least one selected from the group consisting of titanium oxide, calcium phosphate, hydroxyapatite, alumina, aluminum hydroxide, silver, gold, silica, and polystyrene, on the surface of the biocompatible polymer. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is bound to. The pharmaceutical composition according to claim 11 or 12, wherein the biocompatible polymer is at least one water-soluble polymer selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyethylene oxide, and dextran. The pharmaceutical composition according to claim 12 or 13, wherein the complex is a biocompatible polymer bonded to the surface of particles made of titanium oxide. The pharmaceutical composition according to any one of claims 1 to 14, wherein the chemotherapeutic agent is a DNA synthesis inhibitor, a cell division inhibitor, or a DNA damaging agent. The chemotherapeutic agents include etoposide, cisplatin, oxaliplatin, gemcitabine, methotrexate, leucovorin, pemetrexed disodium, camptothecin, doxorubicin, vinblastine, vincristine, bindesin, citarabin, azathiopurine, melphalan, imatinib, anastrosol , Paclitaxel, docetaxel, vincristine, mitoxantrone, artesunate, capecitabin, etoposide, vinorelbine, and at least one selected from the group consisting of TOPK inhibitors such as OTS964, NLG919, OTS167 and OTSC41, claims 1-15. The pharmaceutical composition according to any one of the above. The chemotherapeutic agent is any one of claims 1 to 16 used in combination with a cancer treatment selected from the group consisting of surgery, photodynamic therapy, radiotherapy, immunotherapeutic agents, cryotherapy and nucleic acid chemotherapeutic agents. The pharmaceutical composition according to the section.

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