Photo-sensitizing pigment

Abstract

The present invention addresses the problem of providing a compound which has absorption in the near-infrared region and achieves a high efficiency of singlet oxygen generation (quantum yield), while exhibiting high tumor toxicity in combination with immunotoxin. The present invention provides a compound represented by formula (1) or a salt thereof. (In the formula, each of L1 and L2 independently represents a single bond, -O-, -CO-, an alkylene group having from 1 to 8 carbon atoms, a sugar chain or a combination thereof; each of R1 and R2 independently represents an alkyl group having from 1 to 8 carbon atoms, a carboxylic acid group, an amino group, a hydroxyl group, a thiol group or a biotin residue; each of R3, R4, R5, R6, R7 and R8 independently represents an alkyl group having from 1 to 8 carbon atoms, a phenyl group, a carboxylic acid group, an amino group, a hydroxyl group, a thiol group or a biotin residue; and M represents Mg, Zn, Fe, P, Si, Cu, Sn, Al, Ti, Mo or Ni.)

Description

Technical Field

[0001] The present invention relates to a novel photosensitizing dye useful for killing tumor cells and its use.

Background Art

[0002] Currently, surgery, anticancer drug therapy, and radiation therapy are the main treatment methods for cancer. However, each treatment method is accompanied by pain and side effects, imposing a heavy burden on the patient's body. Photodynamic therapy has attracted attention as a treatment method for reducing the burden on patients. Photodynamic therapy is a treatment method in which a photosensitizer is administered into the body and accumulated in tumor cells, and near-infrared light is irradiated pinpointedly thereto to generate singlet oxygen, and the cytotoxicity thereof kills the tumor cells. Photodynamic therapy can treat lesions that cannot be surgically removed and has many advantages such as almost no side effects like anticancer drugs, but there are also some disadvantages that hinder its widespread practical use. One is the problem of photosensitizers. Examples of photosensitizers used in photodynamic therapy include Photofrin, Laserphyrin, Indocyanine Green, 5-aminolevulinic acid (5-ALA), etc. However, the absorption maxima of Photofrin (λab = 630 nm), Laserphyrin (λab = 664 nm), and 5-ALA (protoporphyrin IX generated as a result of metabolism in the cytoplasm has λab = 405 nm) are all in the visible light region (Non-Patent Documents 1 and 2), and the biological permeability of visible light is inferior to that of near-infrared light, so it is not suitable for treating lesions deep in the body. Also, although the absorption maximum of Indocyanine Green (λab = 800 nm) is in the near-infrared region, it lacks chemical stability due to having a long unsaturated carbon chain and is decomposed within dozens of seconds by light irradiation and thus does not function as a photosensitizer (Non-Patent Document 3).

[0003] Another drawback is the low tumor cell-specific accumulation of photosensitizers. Porphyrins such as Photofrin and Laserphilin are known to accumulate in tumors, although the exact mechanism is not clear. In addition, studies have been conducted on indocyanine green to increase the molecular size by binding it to liposomes or polymers to form nanoparticles, thereby improving the uptake efficiency in tumors by the EPR effect (Non-Patent Documents 4 and 5). The EPR effect refers to the property that due to the immature blood vessel structure of tumors, macromolecular drugs with a size of several hundred nanometers accumulate, and due to the immaturity of lymphatic tissues, the drugs taken up cannot be excreted outside the tissue, resulting in the accumulation of macromolecular drugs in tumor cells. However, the difference in the concentration of these photosensitizers between tumor cells and normal cells is small, and it cannot be said to have sufficient specificity.

[0004] Although photodynamic therapy still has many problems, in recent years, the development of new treatment methods based on photodynamic therapy has also advanced. One of them is photoimmunotherapy devised by Hisataka Kobayashi et al. of the National Cancer Institute of the United States (Non-Patent Document 6). Photoimmunotherapy is a photodynamic therapy in which a photosensitizer IR700DX is bound to an antibody that binds to a protein specifically expressed on tumor cells. Due to the antigen-antibody reaction, the specific accumulation of the drug in tumor cells is greatly enhanced compared to when the photosensitizer is administered alone. Cancer treatment with antibody drugs has been carried out before. Its mechanism of action can be broadly divided into two. When an antibody binds to a cell or pathogen, immune cells such as macrophages and NK cells that recognize the Fc region of the antibody are attracted, and the ADCC activity of killing the cells or pathogens to which the antibody is bound. And when complement binds to the Fc region of the antibody bound to the target antigen, a cascade of complement activation reactions occurs, and finally the formed complex dissolves the cell membrane, which is called CDC activity. Furthermore, in order to enhance the tumor-killing ability, in addition to these two actions, treatments using antibodies conjugated with anticancer drugs (Non-Patent Document 7) or antibodies labeled with radioactive substances (Non-Patent Document 8) have also been carried out. However, there was a problem that anticancer drugs and radioactive substances also damaged cells other than tumor cells. On the other hand, in photoimmunotherapy, after accumulating the antibody conjugated with the photosensitizer in tumor cells, the site where the drug acts can be doubly limited by irradiating the site with a laser, so it can be said that it is a treatment method with less damage to normal cells and fewer side effects.

[0005] Another one is photochemical internalization (PCI). PCI is a drug delivery method that enhances the endosomal escape efficiency of drugs (mainly biopolymers) through photosensitizers and light irradiation, and was first proposed by Berg et al. in 1999 (Non-Patent Document 9). Usually, after biopolymers such as proteins and nucleic acids are taken up by cells through the endosomal pathway, they mainly migrate to the lysosomal degradation system, so it is difficult for their functions to be fully exerted intracellularly. Therefore, in PCI, singlet oxygen generated by irradiating the photosensitizer with light destroys the endosomal membrane before it migrates to the lysosome, and releases the biopolymer into the cytoplasm. As a result, the biopolymer can function intracellularly without being degraded. In 2016, a clinical trial (phase 1) was conducted to enhance the efficacy of the anticancer drug bleomycin by PCI using the photosensitizer TPCS2a. Complete disappearance of tumors was observed in 58% of the patients, and tumor shrinkage was observed in 11% of the patients (Non-Patent Document 10). Thus, it is suggested that drugs that could not exert their efficacy due to low endosomal escape efficiency in the past may be able to provide sufficient therapeutic effects by PCI. Also, as another method, a technique has been devised that combines a substance that binds to a target substance on the surface of tumor cells, a conjugate of a cytotoxin, and a photosensitizer. According to this technique, a conjugate of a substance that binds to a target substance on the surface of tumor cells (e.g., immunotoxin) and a cytotoxin that has been internalized into the endosome of tumor cells can exert a strong therapeutic effect by endosomal escape upon irradiation of the photosensitizer with light.

[0006] Near-infrared light is light with a wavelength of about 700 to 1500 nm. It has characteristics such as low absorption by water and hemoglobin, low scattering by biomolecules, high biological permeability, low energy and no damage to the living body, and low autofluorescence, and is useful for the observation and control of living bodies. Therefore, near-infrared light-absorbing dyes are used in various imaging (fluorescence, two-photon, photoacoustic wave) and disease treatment (photodynamic therapy, photothermal therapy). However, since there are few types of near-infrared light-absorbing dyes, the scope of application is still narrow.

[0007] Compared with the large number of dyes that absorb ultraviolet and visible light, the types of near-infrared light-absorbing dyes are limited to phthalocyanines, squaraines, and some cyanine dyes. Some dyes are already used clinically for the treatment of diseases, but these dyes have many problems when considering their application to living organisms. Phthalocyanine has the property of low water solubility and easily forming aggregates in water. To solve the problem of low water solubility, many phthalocyanines with sulfonic groups introduced have been developed, but anionic substituents repel the phosphate groups of cell membranes, making it difficult for them to be taken up by cells (Non-Patent Document 11). Squaraine has low water solubility and at the same time has a highly electrophilic cyclobutyl skeleton, so it has high chemical reactivity and may react with biomolecules (Non-Patent Document 12). Cyanine has a small Stokes shift and is easily affected by scattered light, and cyanines with long absorption wavelengths have long unsaturated carbon chains, so they lack chemical stability (Non-Patent Document 13). In addition, functionalization aimed at application to living organisms has not progressed for all dyes, but when functionalization is carried out, the molecular size becomes large, and furthermore, the biocompatibility becomes low. Considering these problems, there is a need to develop dye molecules that are suitable for application to living organisms and can fully exhibit the properties of near-infrared light-absorbing dyes.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0009] As described above, the near-infrared absorbing dyes currently used as photosensitizers have problems such as insufficient absorption wavelength length and lack of chemical stability. An object to be solved by the present invention is to provide a compound that has absorption in the near-infrared region, has high efficiency (quantum yield) of singlet oxygen generation, and has high tumor damaging properties in combination with an immunotoxin.

Means for Solving the Problems

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that by introducing a new substituent and a central metal into the phthalocyanine skeleton, the absorption maximum can be shifted to a longer wavelength, the singlet oxygen quantum yield can be improved, and the therapeutic effect in photodynamic therapy can be enhanced. The present invention has been completed based on these findings.

[0011] According to the present invention, the following inventions are provided. <1> A compound represented by formula (1) or a salt thereof.

Chemical Formula

[0012] <6> A medicament for killing tumor cells, comprising the compound or a salt thereof according to any one of <1> to <5>. <7> (1) A step of contacting the compound or a salt thereof according to any one of <1> to <5> with tumor cells; and (2) A step of killing the cells by irradiating the tumor cells with a wavelength effective for activating the compound or a salt thereof according to any one of <1> to <5>: The medicament according to <6>, which kills tumor cells by the above method. <8> (a) The compound or a salt thereof according to any one of <1> to <5>, and (b) A low molecular weight antitumor agent, A medicament for killing tumor cells, comprising the above components. <9> (1) A step of contacting the compound or a salt thereof according to any one of <1> to <5> and the low molecular weight antitumor agent with tumor cells, and (2) A step of killing the cells by irradiating the tumor cells with a wavelength effective for activating the compound or a salt thereof according to any one of <1> to <5> thereafter: The medicament according to <8>, which kills tumor cells by the above method. <10> (a) The compound or a salt thereof according to any one of <1> to <5>, and (b) A conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent, A medicament for killing tumor cells, comprising the above components. <11> The pharmaceutical according to <10>, wherein the substance that binds to the target substance on the surface of tumor cells is a conjugate of an antibody or a fragment thereof, a ligand or a peptide, and a cytotoxin. <12> The pharmaceutical according to <9>, wherein the antibody is an antibody against Epidermal Growth Factor Receptor (EGFR, ERBB1, ERBB2, ERBB3, ERBB4), Mesothelin, Ephrin type-A receptor 2 (EphA2), Glypican3 (GPC3), Cadherin17 (CDH17), Cadherin3 (CDH3), or Roundabout homolog 1 (Robo1). <13> The pharmaceutical according to any one of <8> to <10>, wherein the cytotoxin is saporin, gelonin, or Pseudomonas aeruginosa exotoxin. <14> (1) A step of contacting a compound according to any one of <1> to <5> or a salt thereof with the conjugate and tumor cells, and (2) A step of killing the cells by irradiating the tumor cells with a wavelength effective for activating the compound according to any one of <1> to <5> or a salt thereof: The pharmaceutical according to any one of <10> to <13>, which kills tumor cells by the above method. <15> The pharmaceutical according to any one of <1> to <14>, wherein the tumor cells are cancer cells of any one of head and neck cancer, lung cancer, liver cancer, colorectal cancer, skin cancer, esophageal cancer, gastric cancer, cervical cancer, endometrial cancer, mesothelioma, brain tumor, malignant melanoma, breast cancer, bile duct cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, or malignant lymphoma, osteosarcoma.

Effect of the Invention

[0013] The compound of the present invention has absorption in the near-infrared region, has a high efficiency (quantum yield) of singlet oxygen generation, and has high tumor damaging ability in combination with immunotoxin.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail. <Compound of the Present Invention> The present invention relates to a compound represented by formula (1) or a salt thereof.

Chemical formula

[0016] The compound of the present invention has a thio group, which is an electron-donating group for destabilizing the energy level of the HOMO and increasing the absorption wavelength, at the α-position. Since the α-position of phthalocyanine has a larger molecular orbital coefficient than the β-position, by introducing an electron-donating group at the α-position, the energy level of the HOMO can be destabilized, and the absorption wavelength of phthalocyanine can be shifted to the longer wavelength side.

[0017] The compound of the present invention or a salt thereof is a photosensitizer that induces photochemical intracellular translocation upon activation by light and can generate singlet oxygen upon activation by light.

[0018] L 1 and L 2 each independently represents a single bond, -O-, -CO-, an alkylene group having 1 to 8 carbon atoms, a sugar chain, or a combination thereof. L 1 and L 2 may be, for example, -OCO- which is a combination of -O- and -CO-, or -COO-, or a group formed by combining one or more of -O-, -CO-, -OCO-, or -COO- with one or more alkylene groups having 1 to 8 carbon atoms. L 1and L 2 is preferably an alkylene group having 1 to 8 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, and still more preferably an alkylene group having 1 to 4 carbon atoms.

[0019] R 1 and R 2 each independently represents an alkyl group having 1 to 8 carbon atoms, a carboxylic acid group, an amino group, a hydroxyl group, a thiol group, or a biotin residue. R 1 and R 2 is preferably a carboxylic acid group.

[0020] R 3 、R 4 、R 5 、R 6 、R 7 and R 8 each independently represents an alkyl group having 1 to 8 carbon atoms, a phenyl group, a carboxylic acid group, an amino group, a hydroxyl group, a thiol group, or a biotin residue. R 3 、R 4 、R 5 、R 6 、R 7 and R 8 are each independent, that is, they may be the same or different, but preferably R 3 、R 4 、R 5 、R 6 、R 7 and R 8 are the same group. R 3 、R 4 、R 5 、R 6 、R 7 and R 8 is preferably a phenyl group.

[0021] M represents Mg, Zn, Fe, P, Si, Cu, Sn, Al, Ti, Mo or Ni. M particularly preferably represents Zn.

[0022] The synthesis method of the compound of the present invention is not particularly limited and can be synthesized by a method according to the examples described below. Depending on the type of substituents in formula (1), the compound of formula (1) can be synthesized using the corresponding reagents.

[0023] <Pharmaceutical for killing tumor cells> The pharmaceutical of the present invention includes the following three forms. The first form is a pharmaceutical for killing tumor cells, which contains the compound represented by formula (1) or a salt thereof. In this form, the tumor cells can be killed by (1) a step of contacting the compound represented by formula (1) or a salt thereof with the tumor cells; and (2) a step of irradiating the tumor cells with a wavelength effective for activating the compound represented by formula (1) or a salt thereof to kill the cells.

[0024] The second form is a pharmaceutical for killing tumor cells, which contains (a) the compound represented by formula (1) or a salt thereof and (b) a low-molecular-weight antitumor agent. In this form, the tumor cells can be killed by (1) a step of contacting the compound represented by formula (1) or a salt thereof and the low-molecular-weight antitumor agent with the tumor cells, and (2) a step of irradiating the tumor cells with a wavelength effective for activating the compound represented by formula (1) or a salt thereof to kill the cells. In the present invention, the low-molecular-weight antitumor agent is encapsulated in endosomes after binding to the tumor. The compound (photosensitizing dye) represented by formula (1) added separately (or simultaneously) is irradiated with light to release the low-molecular-weight antitumor agent in the endosomes into the cytoplasm. The tumor cells can be killed.

[0025] The order of contacting the compound represented by the formula (1) or a salt thereof with the low-molecular-weight antitumor agent with tumor cells is not particularly limited. After administering the compound represented by the formula (1) or a salt thereof, the low-molecular-weight antitumor agent may be administered, or after administering the low-molecular-weight antitumor agent, the compound represented by the formula (1) or a salt thereof may be administered, or the compound represented by the formula (1) or a salt thereof and the low-molecular-weight antitumor agent may be administered simultaneously.

[0026] The third form is a medicament for killing tumor cells, which comprises (a) a compound represented by the formula (1) or a salt thereof, and (b) a conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent. In this form, tumor cells can be killed by (1) a step of contacting the compound represented by the formula (1) or a salt thereof and the conjugate with tumor cells, and (2) a step of irradiating the tumor cells with a wavelength effective for activating the compound represented by the formula (1) or a salt thereof to kill the cells. In the present invention, a conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent is encapsulated in an endosome after binding to a tumor. By irradiating light on the compound (photosensitizing dye) represented by the formula (1) added separately (or simultaneously), the above conjugate (immunotoxin) (or its degradation product) in the endosome is released into the cytoplasm to kill tumor cells.

[0027] The order of contacting the compound represented by the formula (1) or a salt thereof with the conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent with tumor cells is not particularly limited. After administering the compound represented by the formula (1) or a salt thereof, the above conjugate may be administered, or after administering the above conjugate, the compound represented by the formula (1) or a salt thereof may be administered, or the compound represented by the formula (1) or a salt thereof and the above conjugate may be administered simultaneously.

[0028] <Low-molecular-weight antitumor agent> A preferred example of the anticancer agent used in the present invention is bleomycin. Other preferred examples include anticancer antibiotics, alkylating agents, platinum compounds, antimetabolites, topoisomerase inhibitors, anticancer antibiotics, microtubule-acting anticancer agents (alkaloid anticancer agents), molecular target drugs (kinase inhibitors, etc.), immunomodulatory drugs, DNA intercalating agents or DNA cross-linking agents, DNA synthesis inhibitors, DNA and / or RNA transcription inhibitors, and the like.

[0029] <Substance that binds to a target substance on the surface of tumor cells> Examples of the substance that binds to a target substance on the surface of tumor cells include, but are not particularly limited to, antibodies or fragments thereof, ligands, or peptides.

[0030] When using an antibody as the substance that binds to a target substance on the surface of tumor cells, an antibody that specifically binds to a target substance on the surface of tumor cells (for example, proteins such as Epidermal Growth Factor Receptor (EGFR, ERBB1, ERBB2, ERBB3, ERBB4), Mesothelin, Ephrin type-A receptor 2 (EphA2), Glypican3 (GPC3), Cadhelin17 (CDH17), Cadherin3 (CDH3), Roundabout homolog 1 (Robo1), CD20, etc.) can be used.

[0031] The type of antibody is not particularly limited, and it may be any of a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a sheep antibody, a camel antibody, a chicken antibody, etc., or a genetically engineered antibody artificially modified for the purpose of reducing the heterologous antigenicity against humans, such as a chimeric antibody, a humanized antibody, etc. The genetically engineered antibody can be produced using known methods. A chimeric antibody is an antibody consisting of the variable regions of the heavy and light chains of a non-human mammal, for example, a mouse antibody, and the constant regions of the heavy and light chains of a human antibody. It can be obtained by ligating the DNA encoding the variable region of the mouse antibody with the DNA encoding the constant region of the human antibody, incorporating this into an expression vector, and introducing it into a host for production. A humanized antibody is one in which the complementarity-determining regions (CDRs) of a non-human mammal, for example, a mouse antibody, are transplanted into the complementarity-determining regions of a human antibody, and its general genetic engineering techniques are also known. Specifically, a DNA sequence designed to ligate the CDR of the mouse antibody with the framework region (FR) of the human antibody is synthesized by the PCR method from several oligonucleotides prepared to have overlapping portions at the ends. The obtained DNA is ligated with the DNA encoding the human antibody constant region, then incorporated into an expression vector, and introduced into a host for production (Japanese Patent Application Laid-Open No. 239400, International Publication WO96 / 02576, etc.).

[0032] Methods for obtaining human antibodies are also known. For example, human lymphocytes can be sensitized in vitro with a desired antigen or cells expressing a desired antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266, to obtain a desired human antibody having binding activity to the antigen (see Japanese Patent Publication No. 1-59878). Also, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO94 / 25585, WO96 / 34096, WO96 / 33735). Furthermore, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the variable region of a human antibody can be expressed on the surface of a phage by the phage display method as a single-chain antibody (scFv), and phages that bind to an antigen can be selected. By analyzing the gene of the selected phage, a DNA sequence encoding the variable region of a human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an appropriate expression vector with the sequence. These methods are already well-known, and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388.

[0033] The antibody that binds to tumor cells is preferably a humanized or human antibody, but is not limited thereto.

[0034] In addition, as long as these antibodies do not lose the property of recognizing the full length or a part of the protein encoded by the antigen gene on the tumor cell surface, they may be low-molecular-weight antibodies such as antibody fragments (fragments) or modified antibodies. An antibody fragment is a part of an antibody that retains the ability to bind to ROBO1. Specific examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, Fv, Diabody, single-chain antibody fragment (scFv), and the like. To obtain such antibody fragments, a gene encoding these antibody fragments may be constructed, introduced into an expression vector, and then expressed in an appropriate host cell. As a modified antibody, an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.

[0035] DNA encoding a monoclonal antibody can be easily isolated and sequenced by a conventional method (for example, using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells are a preferred starting material for such DNA. Once isolated, the DNA can be inserted into an expression vector, recombined into host cells such as E. coli cells, COS cells, CHO cells, or myeloma cells that do not produce immunoglobulins unless transformed, and monoclonal antibodies can be produced from the recombinant host cells.

[0036] A ligand can be used as a substance that binds to the target substance on the tumor cell surface. When the target substance on the tumor cell surface is a receptor such as, for example, Epidermal Growth Factor Receptor (EGFR, ERBB1, ERBB2, ERBB3, ERBB4), Mesothelin, Ephrin type-A receptor 2 (EphA2), etc., a ligand for the above receptor can be used.

[0037] As the target of the antibody, epidermal growth factor receptor (EGFR) was used. EGFR is a receptor that recognizes epidermal growth factor involved in cell proliferation and growth and conducts signal transduction. Although EGFR is expressed at about 40,000 to 100,000 molecules per cell even in normal cells

[15] , expression of more than 1,000,000 molecules per cell has been confirmed in many cancers such as breast cancer, bladder cancer, colon cancer, glioma, non-small cell lung cancer, pancreatic cancer, ovarian cancer, and head and neck cancer

[16] . In addition, tumors with overexpression of EGFR show higher proliferative ability and metastatic ability compared to tumors without overexpression, and it has also been reported that they are resistant to conventional chemotherapy and radiotherapy

[17] . Therefore, EGFR has already become a common target as a target for cancer treatment. Gefitinib (Iressa), an EGFR tyrosine kinase inhibitor, inhibits autophosphorylation of EGFR and blocks signal transduction by competitively binding to the ATP binding site of the tyrosine kinase of EGFR with ATP

[18] . Gefitinib was also approved in Japan in 2002 and is used for the treatment of non-small cell lung cancer. In addition, the anti-EGFR antibody drugs cetuximab and panitumumab were also approved in Japan in 2008 and 2010, respectively, and are used for clinical treatment. Thus, EGFR, which also serves as a target for molecular target drugs in clinical practice, was selected as the target of a general antibody.

[0038] As a substance that binds to the target substance on the tumor cell surface, a peptide can also be used. A peptide that binds to the target substance on the tumor cell surface can be designed and manufactured by those skilled in the art.

[0039] <Cytotoxin> For the cytotoxin, a protein having cytotoxicity is preferred, but it is not limited thereto, and it may be a synthetic or natural anti-cancer compound such as bleomycin or a compound used in ADC. Preferred embodiments of the cytotoxic protein include saporin, gelonin, Pseudomonas aeruginosa exotoxin, ricin A chain, deglycosylated ricin A chain, ribosome-inactivating protein, alpha-sarcin, aspergillin, restrictocin, ribonuclease, epipodophyllotoxin, diphtheria toxin, shiga toxin and its variants, and recombinant genes. In addition, an anticancer agent that is difficult to translocate from the endosome into the cytoplasm can also be used.

[0040] A preferred example of the cytotoxin is saporin. Saporin is a proteinaceous toxin contained in the seeds of Saponaria officinalis, which causes cell death by inactivating ribosomes. Saporin alone can only be taken up by cells by passive methods such as pinocytosis, and the cell uptake efficiency is low. Therefore, it is known that saporin can only be taken up by cells and exert its toxicity after being formed into an immunotoxin bound to an antibody.

[0041] <Conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxin> The substance that binds to the target substance on the surface of tumor cells and the cytotoxin must be bound directly or indirectly. When using an antibody or a fragment thereof as the substance that binds to the target substance on the surface of tumor cells, the method of directly chemically conjugating this with the cytotoxin can use the conjugation method used in known ADC (Antibody Drug Conjugate). Also, when the cytotoxin is a protein, it is also possible to use a bifunctional cross-linking agent.

[0042] Also, when the cytotoxin is a protein, it is also possible to create an immunotoxin by making a protein in which the toxin is recombinantly fused with an antibody or a fragment thereof. As another method, it is also possible to use a technique of indirectly conjugating an antibody or a fragment thereof and a cytotoxin using a second binding pair. Examples of the second binding pair include the use of avidin-biotin, antibody-hapten, etc.

[0043] In the present invention, instead of an immunotoxin in which an antibody and a toxin are conjugated, it is also possible to use a conjugate of a peptide or ligand that binds to a target substance on the surface of tumor cells and a toxin.

[0044] <Administration Method and Dosage> The administration method when administering the pharmaceutical of the present invention to a subject having a tumor (such as cancer) is not particularly limited. The compound represented by formula (1) of the present invention or a salt thereof can be administered, for example, by intravenous administration, arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, or oral administration. There are also methods such as local injection, application, spraying, etc. in the tumor tissue and its periphery. The low-molecular anti-tumor agent can be administered, for example, by intravenous administration, arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, or oral administration. There are also methods such as local injection, application, spraying, etc. in the tumor tissue and its periphery. The conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent can be administered, for example, by intravenous administration, arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, or oral administration. There are also methods such as local injection, application, spraying, etc. in the tumor tissue and its periphery.

[0045] The dosage of the compound represented by formula (1) of the present invention or a salt thereof is not particularly limited, but can be administered, for example, at 1 μg / kg body weight to 100 mg / kg body weight, preferably 10 μg / kg body weight to 10 mg / kg body weight. The dosage of the low-molecular anti-tumor agent is not particularly limited, but can be administered, for example, at 1 μg / kg body weight to 100 mg / kg body weight, preferably 10 μg / kg body weight to 10 mg / kg body weight. The dosage of the conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent is not particularly limited, but can be administered, for example, at 1 μg / kg body weight to 100 mg / kg body weight, preferably 10 μg / kg body weight to 10 mg / kg body weight.

[0046] The number of administrations is not particularly limited and can be carried out one or more times (from 1 to 20 times, preferably from 1 to 10 times), for example, it can be carried out every 2 - 4 weeks or every 1 - 2 months. Also, the number of light irradiations is not particularly limited and can be carried out one or more times.

[0047] <Target tumor> The tumors targeted for administration of the medicament of the present invention are not particularly limited, and examples include head and neck cancer, lung cancer, liver cancer, colorectal cancer, skin cancer, esophageal cancer, gastric cancer, cervical cancer, endometrial cancer, mesothelioma, brain tumor, malignant melanoma, breast cancer, bile duct cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer or malignant lymphoma, osteosarcoma and other cancers.

[0048] Tumor cells are preferably tumors expressing Epidermal Growth Factor Receptor (EGFR, ERBB1, ERBB2, ERBB3, ERBB4), Mesothelin, Ephrin type - A receptor 2 (EphA2), Glypican3 (GPC3), Cadhelin17 (CDH17), Cadhelin3 (CDH3), or Roundabout homolog 1 (Robo1), CD20, etc. on the surface.

[0049] The present invention will be specifically described by the following examples, but the present invention is not limited by the examples.

Examples

[0050] <Synthesis of phthalonitrile Pn3>

Chem.

[0051] 2,3 - Dicyano hydroquinone (1.0 g, 6.24 mmol), ethyl 4 - bromobutyrate (3.57 mL, 24.5 mmol), and potassium hydroxide were dissolved in dimethyl sulfoxide (30 mL) and stirred at room temperature for 18 h. Water was added, and the resulting precipitate was collected, washed with water, and dried under reduced pressure to obtain the target product (2.12 g, 88%). Since it is a known compound, NMR assignments were made according to the literature (T. Goslinski et al., Polyhedron, 2011, 30, 1538 - 1546).

[0052] 1H NMR (DMSO d6) δ 7.62 (s, 2H), 4.18(t, J = 6.0, 4H), 4.07 (q, J = 7.1, 4H), 2.38 (t, 2H), 1.98 (qui, J = 6.9, 4H),1.78 (t, J = 6.9, 6H); ESI - MS (positive, smart) [M + Na]+ 411.1527(found), 411.1527(calcd)

[0053] <Synthesis of phthalonitrile Pn4>

Chemical Structure

[0054] 1H NMR (DMSO d6) δ 7.81(d, J = 4.1, 4H), 7.75(s, 2H), 7.56 (d, J = 4.1, 4H), 2.47 (s, 6H); ESI-MS (pos.smart) [M+Na]+ 491.0346 (found), 491.0342 (calcd)

[0055] <Synthesis of phthalonitrile Pn5>

Chemical formula

[0056] 1H NMR (CDCl3) δ 7.41-7.49 (m, 10H), 6.95(s, 2H); ESI-MS (pos. smart) [M+Na]+ 367.0334 (found),367.0334 (calcd)

[0057] <Synthesis of phthalocyanine H2Pc6>

Chemical formula

[0058] MALDI-TOF-MS (LP_0-2kDa) [M+H]+ 1368.453 (found), 1368.689 (calcd)

[0059] <Synthesis of zinc phthalocyanine PS1>

Chemical formula

[0060] 1H NMR (DMSO d6) δ 7.91 (d, J = 6.6, 4H), 7.75 (m, 12H), 7.65 (m, 6H), 7.51 (m, 12H), 7.20 (d, J = 7.4, 2H), 7.06 (d, J = 9.2, 2H), MALDI-TOF-MS (LP_0-2kDa) [M+H]+ 1432.309 (found), 1432.054 (calcd)

[0061] <Measurement of optical properties of ZnPc6> The absorption spectrum of the synthesized PS1 was measured. The absorption spectrum of PS1 in dimethyl sulfoxide is as shown in Fig. 1, and the maximum absorption wavelength in the Q band was 775 nm. Also, absorption spectra at different concentrations were measured, the absorption at a wavelength of 775 nm was plotted, and when the molar absorption coefficient was determined, it was 41000(M -1 cm -1 ) (Fig. 2).

[0062] <Measurement of the singlet oxygen quantum yield of PS1> The singlet oxygen quantum yield of PS1 was measured. The results of measuring the absorption spectrum every 5 seconds of irradiating light with a wavelength of 775 nm after dissolving PS1 and DPBF in dimethyl sulfoxide are as shown in Fig. 3. From these results, it was confirmed that the absorption at a wavelength of 416 nm decreased as the light irradiation time increased, indicating that DPBF was reacting with singlet oxygen. As a result of calculating the singlet oxygen quantum yield, it was 0.61. This value is higher than that of conventional photosensitizers, and it is a result that can be expected to be useful for photodynamic therapy.

[0063] <Cytotoxicity of PS1 and near-infrared light irradiation> To confirm the ability of PS1 to release singlet oxygen in cells, a cell experiment was conducted. First, A431 (10,000 cells / well) was seeded in a 96-well plate, and after 24 hours, PS1 (0 - 20 μM) was added. After 18 hours, the medium containing PS1 was removed, the cells were washed with PBS, fresh medium was added, and near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. 48 hours later, CCK-8 was added, and the cell viability was measured by measuring the absorption at a wavelength of 450 nm with a plate reader (Fig. 4). When PS1 was at 4 μM or less, no decrease in cell viability was observed regardless of the presence or absence of near-infrared light irradiation, but at 20 μM, the cell viability significantly decreased only under near-infrared light irradiation. From these results, it was confirmed that PS1 can release singlet oxygen in the cell environment by near-infrared light irradiation.

[0064] <Evaluation of the Cellular Retention of PS1> The cellular retention of PS1 was evaluated. If PS1 remains in the body without being excreted from cells, patients administered with PS1 have to stay in a dark room until PS1 is completely excreted from the body in order to avoid damage to organs such as the skin by singlet oxygen at unwanted sites due to photosensitization. Therefore, being quickly excreted outside the body is also one of the properties that a photosensitizer should possess. To evaluate the cellular retention of PS1, the following experiment was conducted (S. Hirohara et al., J. Photochem. Photobiol., B, 2005, 78, 7-15).

[0065] A431 (50,000 cells / well) was seeded in a 24-well plate, and after 24 hours, PS1 (10 μM) was added. After 18 hours, the medium containing PS1 was removed, the cells were washed with PBS, fresh medium was added, and incubation was continued (0, 1, 2, 4, 24 hours). After washing the cells with PBS again, DMSO (150 μL) was added and shaken for 1 hour. Then, the absorption spectrum of the obtained DMSO solution was measured to quantify the PS1 remaining in the cells (Figure 5). As a result, it was found that 52% of PS1 was released extracellularly after incubation for 24 hours excluding PS1 (Figure 6). Since actual cells exist in a more fluid environment, it is considered that the excretion of PS1 is faster than when cells are cultured in a culture dish.

[0066] <Biotinylation of the Anti-EGFR Antibody Panitumumab> To prepare an immunotoxin targeting EGFR, biotinylation of the anti-EGFR antibody panitumumab was performed. As a method for biotinylating panitumumab, the more efficient NHS ester method was adopted. 40 equivalents of sulfo-NHS-biotin (10 mM, 1.33 μL) was added to panitumumab (1 mg / mL, 50 μL), and the reaction was carried out at room temperature for 1 hour. The reaction solution was purified by spin column chromatography to obtain the biotinylated antibody. Whether the biotinylation of the antibody was progressing was confirmed by ELISA. Panitumumab-biotin or panitumumab was immobilized on a 96-well plate, streptavidin-polyHRP was reacted as the primary antibody, and detection was performed by the enzymatic reaction of tetramethylbenzidine. The result of adding streptavidin-polyHRP was as shown in Figure 7. Since only panitumumab-biotin reacted, it was confirmed that the biotinylation of panitumumab was progressing. An immunotoxin (panitumumab-saporin) was obtained by mixing this biotinylated antibody with 1 equivalent of streptavidin-saporin.

[0067] <Cell experiment using EGFR-overexpressing cells A431> Cell experiments were conducted using A431, which has been confirmed to highly overexpress EGFR. As described for the cytotoxicity of PS1 and near-infrared light irradiation, it was confirmed that PS1 exhibited cytotoxicity only under near-infrared light irradiation at a concentration of 20 μM. Therefore, in photochemical internalization (PCI), the concentrations of PS1 were set at 1 μM and 5 μM, which do not show cytotoxicity with only PS1 and near-infrared light irradiation. First, A431 (10,000 cells / well) was seeded in a 96-well plate, and after 24 hours, immunotoxin (panitumumab-saporin, 0 - 4 nM) and PS1 (1 μM or 5 μM) were added. After 18 hours, the medium containing immunotoxin and PS1 was removed, the cells were washed with PBS, fresh medium was added, and near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. Forty-eight hours later, CCK-8 was added, and cell viability was measured by measuring the absorption at a wavelength of 450 nm using a plate reader (Figure 8). As a result, no decrease in cell viability was observed with the immunotoxin alone at 160 pM or less, while when combined with PCI, the cell viability significantly decreased even with 6.4 pM of the immunotoxin. Also, when only PCI was performed without adding the immunotoxin (IT 0 pM in Figure 8), no significant decrease in cell viability was observed. From these results, it was found that PCI using PS1 can significantly enhance the cytotoxic ability of the immunotoxin.

[0068] Next, cell experiments were conducted by changing the conditions of the presence or absence of PS1 addition and the presence or absence of near-infrared light irradiation. The results were as shown in Figure 9, and a significant decrease in cell viability was observed only in the cells that underwent both PS1 addition and near-infrared light irradiation in addition to the immunotoxin. From these results, it was suggested that singlet oxygen generated by PS1 and near-infrared light irradiation may contribute to the improvement of the cell-killing ability of the immunotoxin.

[0069] Next, a similar experiment was conducted using a non-specific immunotoxin prepared from B8109B-biotin. The results were as shown in Fig. 10. When the non-specific immunotoxin was used, no significant decrease in cell viability was observed even after the addition of PS1 and near-infrared light irradiation. From these results, it was found that the immunotoxin was taken up by cells having a specific antigen, and its cell-killing ability was enhanced by PCI.

[0070] Next, the photosensitizing ability of PS1 under near-infrared light irradiation was compared with that of conventional photosensitizers. As the conventional photosensitizers, AlPcS2a, indocyanine green, and zinc phthalocyanine were used (Fig. 11). AlPcS2a has been used in many studies on photochemical internalization, but its absorption maximum is in the visible light region and its singlet oxygen quantum yield is as low as 0.17. Indocyanine green (ICG) is a photosensitizer used in photodynamic therapy and photoacoustic imaging, etc., and has an absorption maximum in the near-infrared region, but its singlet oxygen quantum yield is as low as 0.12. Zinc phthalocyanine (ZnPc) is an unsubstituted phthalocyanine having zinc at the center like PS1, and its absorption maximum is in the visible light region, but it has a high singlet oxygen quantum yield.

[0071] Using these photosensitizers, a cell experiment combining the addition of immunotoxin and PCI was conducted. As a result, a significant decrease in cell viability was observed only in the cells subjected to PCI using PS1 (Fig. 12). This is considered to be because AlPcS2a and ZnPc have absorption maxima in the visible light region and thus cannot be photosensitized with near-infrared light, and ICG having an absorption maximum in the near-infrared region cannot release a sufficient amount of singlet oxygen due to its low singlet oxygen quantum yield. From these results, it can be said that PS1 is a better photosensitizer than the conventional ones under near-infrared light irradiation.

[0072] <Verification of Photochemical Internalization (PCI)> From the experiments so far, it was possible to improve the cytotoxicity of the immunotoxin by combining PS1 and near-infrared light irradiation.

[0073] The currently proposed mechanism of PCI is as follows. 1) Both the immunotoxin and the photosensitizer are taken up into cells by endocytosis. 2) By irradiating near-infrared light, singlet oxygen is generated by the action of the photosensitizer, and the endosomal membrane is damaged. 3) The immunotoxin is released into the cytoplasm.

[0074] To prove that PCI is occurring, it is sufficient to observe that the immunotoxin and PS1 are present in the endosome simultaneously and that the immunotoxin is released into the cytoplasm after light irradiation. However, since the absorption maximum of PS1 is at 775 nm in the near-infrared region and it is not suitable for excitation with the laser (633 nm) of a confocal microscope, and the fluorescence quantum yield of PS1 is as low as 0.04, it is considered difficult to observe PS1 in cells. Therefore, we decided to observe the release of the immunotoxin into the cytoplasm by light irradiation. To observe the release of the immunotoxin into the cytoplasm, a fluorescein dye was used. Fluorescein has the property that its fluorescence intensity increases under acidic conditions (M. M. Martin and L. Lindqvist, J. Lumin., 1975, 10, 381 - 390). Utilizing this property, the group of Dr. Otsuki et al. demonstrated the endosomal escape of shRNA labeled with fluorescein and speculated that singlet oxygen generated by near-infrared light irradiation destabilized the endosomal membrane structure, causing an increase in pH within the endosome and an increase in the fluorescence intensity of fluorescein (T. Otsuki et al., Sci. Rep., 2015, 5, 18577). Similar to this experiment, we labeled the antibody with fluorescein and decided to observe the endosomal escape of the antibody due to PCI using a confocal microscope.

[0075] First, a fluorescein-labeled antibody was obtained by mixing biotinylated anti-EGFR antibody with 5 equivalents of streptavidin-FITC. Fluorescein-labeled antibody (1.2 nM) and PS1 (1 μM) were added to A431 (60,000 cells / dish), incubated for 24 hours, then Hoechst33342 (75 nM) was added and incubated for 30 minutes, and the medium was removed. After washing 4 times with PBS, medium without phenol red was added, and observation was performed with a confocal microscope. The results were as shown in Figure 13. Before near-infrared light irradiation, regardless of the presence or absence of PS1, the fluorescence intensity of green fluorescein in the cells was not strong. The places where the fluorescence of fluorescein was strong in dots were considered to be where endosomal escape had already occurred.

[0076] Next, after near-infrared light irradiation (13.8 mW / cm2 for 20 min, 16.6 J / cm2), observation was performed again with a confocal microscope. The results were as shown in Figure 14. An increase in the fluorescence intensity of fluorescein was observed only in the cells to which PS1 was added.

[0077] To quantitatively evaluate the fluorescence intensity, ImageJ was used to quantify the fluorescence of fluorescein per cell. The average value of the green fluorescence derived from fluorescein in the area of the cell was obtained and divided by the number of cells contained in that area to obtain the fluorescence intensity per cell. The number of cells was estimated from the number of nuclei stained with Hoechst33342. The results of quantifying the fluorescence of fluorescein were as shown in Figure 15. From these results, a significant increase in fluorescence intensity was observed only in the cells to which PS1 was added and near-infrared light was irradiated. Therefore, it is considered that endosomal escape of the fluorescein-labeled antibody occurred by PCI using PS1.

[0078] <Cell experiment using EGFR-expressing cell A549> Similar experiments were conducted using A549 cells with low EGFR expression. A549 is a human alveolar basal epithelial adenocarcinoma cell, and it has been reported that the EGFR expression level is approximately 10% of that of A431 (S. Derer et al., J. Immunol., 2012, 189, 5230 - 5239). There are also studies using immunotoxins targeting EGFR on A549 cells (C. Deng et al., Oncotarget, 2017, 8, 38568 - 38580, and X. Zhou et al., J. Cancer Res. Clin. Oncol., 2012, 138, 1081 - 1090), but due to the low EGFR expression level, there are few research examples that could show sufficient cytotoxicity.

[0079] To examine the concentration of PS1 that has no cytotoxicity with only PCI, only PS1 and near - infrared light irradiation were applied to A549. First, A549 (5,000 cell / well) was seeded in a 96 - well plate, and after 24 hours, PS1 (0 - 20 μM) was added. After 18 hours, the medium containing PS1 was removed, the cells were washed with PBS, fresh medium was added, and near - infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. 48 hours later, CCK - 8 was added, and the cell viability was measured by measuring the absorption at a wavelength of 450 nm with a plate reader (Figure 16). As a result, it was found that PS1 below 20 μM has no cytotoxicity regardless of the presence or absence of near - infrared light irradiation. Therefore, the optimal PS1 concentration that has no cytotoxicity with only PCI is considered to be about 1 μM.

[0080] Next, experiments on PCI were conducted on A549 using immunotoxin and PS1. A549 (5,000 cells / well) was seeded in 96-well plates, and after 24 hours, immunotoxin (panitumumab-saporin, 0 - 4 nM) and PS1 (1 μM) were added. After 18 hours, the medium containing immunotoxin and PS1 was removed, the cells were washed with PBS, fresh medium was added, and near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. 48 hours later, CCK-8 was added, and cell viability was measured by measuring the absorption at a wavelength of 450 nm using a plate reader. The results were as shown in Figure 17. By combining PCI, the cytotoxicity of the immunotoxin was significantly improved. Also, no decrease in cell viability was observed with only PCI using 1 μM of PS1. From these results, it was suggested that immunotoxins targeting membrane proteins, which were not previously targeted due to low expression levels, might also acquire cytotoxicity by PCI.

[0081] <Cell experiments using EGFR non-expressing cells HEK293T> Next, similar experiments were conducted using HEK293T cells, which are EGFR non-expressing cells. A549 is a cell derived from human fetal kidney cells and has been reported to have almost no expression of EGFR. First, to examine the concentration of PS1 that has no cytotoxicity with only PCI, only PS1 and near-infrared light irradiation were performed on HEK293T.

[0082] First, HEK293T (10,000 cells / well) was seeded in a 96-well plate, and after 24 hours, PS1 (0 - 20 μM) was added. After 18 hours, the medium containing PS1 was removed, the cells were washed with PBS, fresh medium was added, and near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. 48 hours later, CCK-8 was added, and cell viability was measured by measuring the absorption at a wavelength of 450 nm with a plate reader. The results were as shown in Figure 18, and it was found that PS1 exhibited cytotoxicity by near-infrared light irradiation at a concentration of 0.8 μM or higher. Also, 20 μM of PS1 had strong cytotoxicity regardless of the presence or absence of near-infrared light irradiation. This is thought to be due to the property that HEK293T is easily transfected and takes up more PS1 molecules than other cancer cells. Therefore, the optimal PS1 concentration that does not have cytotoxicity only with PCI is considered to be about 0.1 μM.

[0083] After examining the optimal concentration of PS1, an experiment on PCI was conducted on HEK293T using an immunotoxin and PS1. HEK293T (10,000 cells / well) was seeded in a 96-well plate, and after 24 hours, an immunotoxin (panitumumab-saporin, 0 - 4 nM) and PS1 (0.1 μM) were added. After 18 hours, the medium containing the immunotoxin and PS1 was removed, the cells were washed with PBS, fresh medium was added, and near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm2 for 60 min, 22 J / cm2) was performed. 48 hours later, CCK-8 was added, and cell viability was measured by measuring the absorption at a wavelength of 450 nm with a plate reader. The results were as shown in Figure 19, and no clear decrease in cell viability was observed either with the immunotoxin alone or in combination with PCI. From these results, it was found that the anti-EGFR immunotoxin exhibits cytotoxicity only to cells expressing EGFR even when combined with PCI.

[0084] <Measurement of the membrane damage ability of PS1(Zn6PTPc)> To confirm whether lipid membranes can be destroyed by irradiating PS1(Zn6PTPc) with near-infrared light, a hemolysis assay using red blood cells was performed. The hemolysis assay is a method for examining the degree of hemolysis by quantifying the amount of hemoglobin released outside red blood cells due to hemolysis.

[0085] First, blood collected from mice was centrifuged at 5000 g for 5 minutes to obtain blood cell components. After repeating the washing of 30 μl of the precipitate with PBS at 5000 g for 4 minutes 5 times, the supernatant was discarded and diluted to 550 μl. Then, 20 μl of the red blood cell solution and 80 μl of the PBS solution containing PS1(Zn6PTPc) (final Zn6PTPc concentrations of 0.1, 1, 10, 100 μM) were added to a 96-well plate. Also, PBS without PS1(Zn6PTPc) was used as the negative control, and milliQ was used as the positive control, respectively. After incubating at 37 °C for 100 minutes, near-infrared light (700 - 1100 nm) irradiation (6.1 mW / cm 2 for 60 min, 22 J / cm 2 ) was performed. Further incubation was carried out at 37 °C for 2 hours, the solution in each well was collected, centrifuged at 5000 g for 4 minutes, and 70 μl of the supernatant was transferred to a new 96-well plate. The amount of hemoglobin released into the supernatant was measured by measuring the absorption at 541 nm with a plate reader. The results were as shown in Figure 20, and it was found that hemolysis was promoted by light irradiation, and the degree increased with the increase in the concentration of PS1(Zn6PTPc). On the other hand, it was found that hemolysis was not induced regardless of the concentration of PS1(Zn6PTPc) when no light was irradiated.

[0086] <Annexin PI cell staining> A431 (100,000 cells / well) was seeded in a 12-well plate, and after 24 hours, immunotoxin (panitumumab-saporin) (160 pM) and PS1 (Zn6PTPc) (0.1, 1, 10 μM) were added. After removing the medium containing immunotoxin and PS1 (Zn6PTPc) 18 hours later, the cells were washed with DMEM, and fresh medium was added followed by near-infrared light (700 - 1100 nm) irradiation (9.9 mW / cm 2 for 37 min, 22 J / cm 2 ) was performed. Forty-eight hours later, staining was carried out according to the protocol of the Annexin V-FITC / PI kit, and the fluorescence intensity was measured by flow cytometry.

[0087] The measurement was performed until the number of cells in the R1 gate reached 10,000, and the results were as shown in Figure 21. As shown in Figure 21(a), as the concentration of PS1 (Zn6PTPc) increased, the number of cells in the gate decreased, and the points near the origin increased. This is presumably because the dead cells were fragmented and could no longer maintain the original cell size. Also, it can be seen from Figure 21(c) that as the concentration of PS1 (Zn6PTPc) increased, the point group moved upward to the right. Since cell death stained with both annexin and PI is apoptosis, in this PCI, as hypothesized, it was shown that saporin induced apoptosis by inactivating ribosomes.

[0088] <Cancer-bearing mouse experiment (A549)> Experiments were conducted using 8 six-week-old nude mice. A549 (4×106 cells / mouse) was transplanted subcutaneously into the right hind leg. When the tumor volume reached 100 mm 3On the day of arrival (Day 0), 8 animals were randomly divided into 4 groups (Group A: control, Group B: PS1(Zn6PTPc) 2.5 mg / kg, Group C: PS1(Zn6PTPc) 5.0 mg / kg, Group D: PS1(Zn6PTPc) 2.5 mg / kg + IT 0.3 mg / kg). PBS was injected into Groups A, B, and C, and immunotoxin (panitumumab-saporin) was prepared at 0.3 mg / kg and injected into Group D, 100 μl each via the tail vein. Two days later (Day 2), PBS was injected into Group A, PS1(Zn6PTPc) 2.5 mg / kg was injected into Groups B and D, and PS1(Zn6PTPc) 5.0 mg / kg was prepared and injected subcutaneously into the tumor, 100 μl each (in serum, containing 22% DMF). Three hours later, near-infrared light (700 - 1100 nm) irradiation (22 J / cm 2 ) was performed.

[0089] The results are shown in Figure 22. The maximum cross-sectional area of the tumor is 5x3 mm. There is no granulation tissue in the tumor. Granulation tissue formation is observed in a thick capsule-like shape at the tumor margin. 50% of the tumor area has fallen into map-like coagulative necrosis. 20% consists of tumor cells that tend to disintegrate, including nuclear condensation and fragmentation, nuclear disintegration, and nuclear fragments. The remaining 30% consists of viable tumor cells.

Claims

1. A compound represented by formula (1) or a salt thereof. 【Chemical 1】 (wherein, L １ and L ２ each independently represents an alkylene group having 1 to 8 carbon atoms, R １ and R ２ represent a carboxylic acid group, R ３ , R ４ , R ５ , R ６ , R ７ and R ８ represent a phenyl group, and M represents Mg, Zn, Fe, P, Si, Cu, Sn, Al, Ti, Mo or Ni)

2. The compound or a salt thereof according to claim 1, wherein M represents Zn.

3. A medicament for killing tumor cells, comprising the compound or a salt thereof according to claim 1 or 2.

4. (1) A step of contacting the compound or a salt thereof according to claim 1 or 2 with tumor cells; and (2) A step of killing the cells by irradiating the tumor cells with a wavelength effective for activating the compound or a salt thereof according to claim 1 or 2: The medicament according to claim 3, which kills tumor cells by the above method.

5. (a) The compound or a salt thereof according to claim 1 or 2, and (b) A low molecular weight antitumor agent, A medicament for killing tumor cells, comprising the above components.

6. (1) A step of contacting the compound or a salt thereof according to claim 1 or 2 and the low molecular weight antitumor agent with tumor cells, and (2) A step of killing the cells by irradiating the tumor cells with a wavelength effective for activating the compound or a salt thereof according to claim 1 or 2: The medicament according to claim 5, which kills tumor cells by the above method.

7. (a) The compound or a salt thereof according to claim 1 or 2, and (b) A conjugate of a substance that binds to a target substance on the surface of tumor cells and a cytotoxic agent, A medicament for killing tumor cells, comprising the above components.

8. The medicament according to claim 7, wherein the substance that binds to the target substance on the surface of tumor cells is an antibody or a fragment thereof, a ligand or a peptide.

9. The medicament according to claim 7, wherein the substance that binds to the target substance on the surface of tumor cells is an antibody against Epidermal Growth Factor Receptor (EGFR, ERBB1, ERBB2, ERBB3, ERBB4), Mesothelin, Ephrin type-A receptor 2 (EphA2), Glypican3 (GPC3), Cadherin17 (CDH17), Cadherin3 (CDH3) or Roundabout homolog 1 (Robo1).

10. The medicament according to any one of claims 7 to 9, wherein the cytotoxic agent is saporin, gelonin, or Pseudomonas aeruginosa exotoxin.

11. (1) A step of contacting the compound or a salt thereof according to claim 1 or 2 and the conjugate with tumor cells, and (2) A step of killing the tumor cells by irradiating the tumor cells with a wavelength effective for activating the compound or a salt thereof according to claim 1 or 2: The pharmaceutical according to any one of claims 7 to 10, which kills tumor cells.

12. The pharmaceutical according to any one of claims 1 to 11, wherein the tumor cells are cancer cells of any one of head and neck cancer, lung cancer, liver cancer, colorectal cancer, skin cancer, esophageal cancer, stomach cancer, cervical cancer, endometrial cancer, mesothelioma, brain tumor, malignant melanoma, breast cancer, bile duct cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer or malignant lymphoma, osteosarcoma.

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