Cancer treatment methods and systems for treating cancer

Abstract

The present disclosure includes a system for treating cancer, the system comprising: an extracorporeal circulation device that is for collecting and returning the blood of a subject, and that includes a blood circuit and a blood pump; and a light irradiation device that is for irradiating the blood circuit with light. A photosensitive substance is administered to the subject before blood collection or is added, before light irradiation, to blood collected from the subject. The blood that has been collected from the subject and has been irradiated with light is returned to the subject. The irradiation energy density of the light is 2.5-300 J / cm2.

Description

[Technical Field]

[0001] This disclosure includes methods for treating cancer, as well as systems for doing so, methods for operating such systems, pharmaceutical compositions, and methods for preparing blood. [Background technology]

[0002] Cancer immunotherapy has recently attracted attention as the fourth treatment method, following surgery, radiation therapy, and drug therapy. Cancer immunotherapy is a treatment method that activates the patient's immune function, and there are two types of treatments: those that strengthen immunity against cancer and those that restore immunity that has been suppressed by cancer. Known cancer immunotherapies include immune checkpoint inhibitors, CAR-T therapy, cytokine therapy, immunostimulants, cancer vaccine therapy, and dendritic cell therapy.

[0003] Photodynamic therapy (PDT) is a treatment method that utilizes photochemical reactions caused by light irradiation and photosensitive substances, and is currently used in the treatment of lung cancer, esophageal cancer, and other cancers. In cancer treatment with PDT, photosensitive substances administered systemically accumulate in cancer cells, thereby damaging cancer while minimizing the impact on normal tissue. Currently, PDT is not used clinically to activate patients' immune functions. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] This disclosure aims to provide a method for treating cancer, as well as a system therefor, a method for operating the system, a pharmaceutical composition, and a method for preparing blood. [Means for solving the problem]

[0005] In one aspect, this disclosure relates to a system for treating cancer, An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The blood collected from the subject and irradiated with light is returned to the subject. The aforementioned light is 2.5~300 J / cm² 2 The light with the irradiation energy density of the system To provide.

[0006] In a further embodiment, the present disclosure relates to a method of operating a system for treating cancer, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light irradiation device emits 2.5 to 300 J / cm². 2 A method for generating light with irradiation energy density. To provide.

[0007] In a further embodiment, the present disclosure relates to a pharmaceutical composition comprising a photosensitive substance for treating cancer, (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light is 2.5~300 J / cm² 2 The method is a method of light with an irradiation energy density. Pharmaceutical composition used To provide.

[0008] In a further embodiment, the present disclosure relates to a method for treating cancer, (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light is 2.5~300 J / cm² 2 The method is a method of light with an irradiation energy density. To provide.

[0009] In a further embodiment, the present disclosure relates to a method for preparing blood for use in the treatment of cancer, Adding a photosensitive substance to blood collected from the subject, and, Irradiating the blood to which a photosensitive substance has been added with light. The blood, which has been irradiated with light, is administered to the target vein, and the light intensity is 2.5 to 300 J / cm². 2 The method is a method of light with an irradiation energy density. To provide. [Effects of the Invention]

[0010] This disclosure provides an effective method for treating cancer, as well as a system therefor, a method for operating the system, a pharmaceutical composition, and a method for preparing blood. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is an explanatory diagram of one embodiment of the present disclosure. [Figure 2] Figure 2 shows the experimental protocol for the example. [Figure 3] Figure 3 shows the tumor volume (left) and tumor tissue excised on day 10 (right) of tumor-bearing rats after subcutaneous transplantation of C6 cells. In the graph on the left, the vertical axis represents tumor volume (mm³), and the horizontal axis represents the number of days after subcutaneous transplantation of C6 cells. [Figure 4]Figure 4 shows IFN-γ production by spleen cells of tumor-bearing rats (left) and tumor-infiltrating CD8+ T cells (right). [Figure 5] Figure 5 shows the tumor volume of tumor-bearing rats after subcutaneous transplantation of C6 cells. The vertical axis represents tumor volume (mm³), and the horizontal axis represents the number of days after subcutaneous transplantation of C6 cells. [Figure 6] Figure 6 shows tumor tissue excised from a tumor-bearing rat on day 10. [Figure 7] Figure 7 shows the staining results of PDT-treated cells and anticancer drug-treated cells with Annexin V and PI. Pre: Untreated cells; 5-ALA: Cells treated with 5-ALA but not with PDT; PDT: Cells immediately after PDT treatment; PDT / 24h: Cells cultured for 24 hours after PDT treatment; MTX, DOX, EPI: Cells cultured for 48 hours after anticancer drug treatment. [Figure 8] Figure 8 shows calreticulin on the cell surface and HMGB1 in the supernatant of PDT-treated cells and anticancer drug-treated cells. [Modes for carrying out the invention]

[0012] Unless otherwise specified, terms used herein have the meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Some definitions of terms used herein are given below, but these definitions take precedence over general understanding.

[0013] In this disclosure, when a number is accompanied by the term "approximately," it is intended to include a range of ±10% of that value. For example, "approximately 20" includes "18 to 22." A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. The "approximately" in relation to a range applies to both endpoints of that range. Therefore, for example, "approximately 20 to 30" includes "18 to 33."

[0014] In this disclosure, cancer is treated using photodynamic therapy (PDT) with a photosensitive substance. It is known that cancer cells are present and circulating in the peripheral blood of cancer patients, not only in patients with hematological cancers but also in patients with solid tumors. When cancer cells in the blood are carried by the bloodstream to organs other than the primary tumor and settle, metastatic lesions occur. When blood collected from a subject who has been administered a photosensitive substance, or blood collected from a subject and to which a photosensitive substance has been added, is irradiated with light, the cancer cells in the blood are damaged. When the blood containing the damaged cancer cells is returned to the subject, the subject's immune response is enhanced, and cancer cells in the subject's body are damaged. In this way, cancer is treated by direct damage to cancer cells by light irradiation and enhancement of the subject's immune response.

[0015] By enhancing the target immune response, cancer cells at any site in the target body, including cancer cells in the light-irradiated blood as well as cancer cells in the primary and metastatic sites, can be damaged, and cancer metastasis can be prevented. In this disclosure, cancer treatment includes treatment of lesions in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs (e.g., lungs, central nervous system, bone, or gastrointestinal tract), skin, or bone marrow. In this disclosure, cancer treatment also includes treatment of the primary site, treatment of metastatic sites (also referred to herein as treatment of cancer metastasis), and prevention of the development of metastatic sites (also referred to herein as prevention of cancer metastasis). In some embodiments, cancer metastasis is prevented or treated. Cancer metastasis includes lesions in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs (e.g., lungs, central nervous system, bone, or gastrointestinal tract), skin, or bone marrow. In further embodiments, cancer metastasis is a skin lesion (e.g., a skin lesion of hematological cancer).

[0016] The cancers in this disclosure are not particularly limited and may be hematological cancers or solid tumors, and may be primary cancers or metastatic cancers. Examples of cancers include adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma, intrahepatic cholangiocarcinoma), kidney cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma. In one embodiment, the cancer is a hematological cancer such as ATL, AML, ALL, CML, or CLL. In a further embodiment, the hematological cancer is ATL. Treatment for ATL includes addressing lesions of the peripheral blood, lymph nodes, spleen, liver or other extranodal organs (e.g., lungs, central nervous system, bone, or gastrointestinal tract), skin, or bone marrow.

[0017] In this specification, "photosensitive substance" means a substance that exerts pharmacological effects upon light irradiation, and includes precursors that produce such substances within cells. Examples of photosensitive substances, though not limited to them, include psoralens, porphyrins, and phthalocyanines, as well as their derivatives, such as ALA compounds, talaporfin sodium, IR700, porfimer sodium, verteporfin, temoporfin, padeliporfin, tetra(methhydroxyphenyl)chlorin (mTHPC), 2-(1-hexyloxyethyl)-2-deninylpyropheophorbide-a (HPPH), ethyl etioplins (SnET2), and hypericin. In the foregoing, "derivative" means a substance that is structurally related to the original substance and possesses the properties of a photosensitive substance. In this specification, the expression "capable of activating photosensitive substances" means that the pharmacological effects of the photosensitive substance can be exerted.

[0018] In one embodiment, the photosensitive substance is an ALA compound. In this specification, an ALA compound means 5-aminolevulinic acid (5-ALA) or its ester or a pharmaceutically acceptable salt thereof. The ALA compound is converted intracellularly to protoporphyrin IX (also referred to herein as PpIX), which selectively accumulates in cancer cells. When PpIX is activated by light irradiation, singlet oxygen is produced, damaging the cancer cells.

[0019] Examples of esters of 5-ALA include 5-ALA-methyl ester (methylaminolevulinate), 5-ALA-ethyl ester, 5-ALA-propyl ester, 5-ALA-hexyl ester (hexaaminolevulinate; HAL), 5-ALA-heptyl ester, and 5-ALA-octyl ester. In some embodiments, the ALA compound is 5-ALA, its methyl or hexyl ester, or a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts include salts with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, or sulfuric acid) or organic acids (e.g., trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or naphthalenedisulfonic acid). In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt or a phosphate salt.

[0020] The photosensitive substance may form a complex with other substances. In one embodiment, the photosensitive substance forms a complex with an antibody that targets cancer cells. The antibody that targets cancer cells is, for example, an antibody against a tumor antigen. Examples of antibodies that target cancer cells include, but are not limited to, antibodies against EGFR, PSMA, CEA, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD147, HER2, VEGF, VEGFR2, CCR4, PD-1, PD-L1, SLAMF7, CTLA4, EpCAM, or PAP2a.

[0021] In one embodiment, the photosensitive substance is administered to the subject prior to blood collection. For example, the photosensitive substance is administered to the subject 2–48 hours, 2–12 hours, 2–10 hours, 2–8 hours, 2–6 hours, 2–4 hours, or 4–6 hours before blood is collected from the subject.

[0022] Photosensitive substances can be administered to subjects orally and via parenteral routes such as intravenously, transdermally, or rectally. In one embodiment, the photosensitive substance is administered orally or intravenously. In a further embodiment, the photosensitive substance is administered orally.

[0023] Photosensitizers are administered in doses that are appropriately adjusted based on factors such as the age and disease of the subject, the photosensitizer used, and the light irradiation energy density. For example, in humans, photosensitizers may be administered at doses of 1 mg to 100 mg / kg, 1 mg to 80 mg / kg, 1 mg to 60 mg / kg, 1 mg to 50 mg / kg, 1 mg to 40 mg / kg, 1 mg to 30 mg / kg, 10 mg to 60 mg / kg, 10 mg to 50 mg / kg, 10 mg to 40 mg / kg, 10 mg to 30 mg / kg, 20 mg to 60 mg / kg, 20 mg to 50 mg / kg, 20 mg to 40 mg / kg, 20 mg to 30 mg / kg, or approximately 20 mg / kg.

[0024] In another embodiment, the photosensitive substance is added to blood collected from the subject prior to light irradiation. For example, the photosensitive substance is added to the blood 1 to 48 hours, 2 to 24 hours, or 4 to 24 hours before light irradiation, for example, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 24 hours before. The amount of the photosensitive substance is added is appropriately adjusted based on factors such as the subject's age and disease, the photosensitive substance used, and the light irradiation energy density. For example, in humans, photosensitive substances may be added in concentrations of 0.001 mg to 1 mg / mL, 0.003 mg to 0.3 mg / mL, or 0.01 mg to 0.3 mg / mL, or 0.01 mmol to 10 mmol / L, 0.03 mmol to 3 mmol / L, 0.03 mmol to 1 mmol / L, 0.1 mmol to 1 mmol / L, 0.25 mmol to 1 mmol / L, 0.3 mmol to 1 mmol / L, or 0.5 mmol to 1 mmol / L.

[0025] In a preferred embodiment, the photosensitive substance is administered to the subject prior to blood collection.

[0026] In this disclosure, subjects may be human or non-human mammals, but are preferably human. Subjects include subjects with cancer and subjects at risk of cancer recurrence, particularly subjects with ATL and subjects at risk of ATL recurrence.

[0027] In this disclosure, the blood in question is collected from a vein and returned to (or administered to) a vein.

[0028] Blood can be collected and returned from the subject using an extracorporeal circulation device, including a blood circuit and a blood pump. The blood circuit is typically connected to the subject's peripheral veins. Commonly used blood circuits and pumps for extracorporeal circulation of blood can be used. The configuration of the blood circuit is not particularly limited as long as extracorporeal circulation is possible, and may include other elements such as a blood bag and a blood component separation device in addition to the blood-carrying tube. If a photosensitive substance is added to the blood collected from the subject, it may be added at any point in the blood circuit, for example, to the blood in the blood bag. In a preferred embodiment, the photosensitive substance is administered to the subject prior to blood collection by the extracorporeal circulation device. The blood circuit should be made of a material that allows the light-irradiated portion to receive light that can activate the photosensitive substance in the blood inside. For example, light is irradiated onto the tube or blood bag within the blood circuit. The length of the irradiated tube or the size of the blood bag, and / or the blood flow rate can be adjusted so that the total light irradiation time to the blood is the desired time. The light may be irradiated onto a portion of the collected blood (for example, the white blood cell differential) or onto the whole blood, but it is preferable to irradiate the whole blood.

[0029] There is no limit to the duration of extracorporeal circulation, but it is typically 1 to 3 hours.

[0030] Blood collection and return may be performed without the use of an extracorporeal circulation device. For example, blood collection and return can be performed using conventional blood collection or return methods such as a blood bag equipped with a syringe or needle (and a blood pump if necessary). In this embodiment, the photosensitive substance may be administered to the subject or added to the blood collected from the subject, but it is preferably added to the collected blood.

[0031] The light irradiation device is not particularly limited as long as it includes a light source that generates light capable of activating a photosensitive material. Examples of light sources include metal halide lamps (Na-Li lamps) containing sodium and lithium, xenon lamps, halogen lamps, LEDs, excimer die lasers, semiconductor lasers, and He-Ne lasers. In one embodiment, the light source is an LED.

[0032] The light may be of a wavelength capable of activating the photosensitive material. The light may be ultraviolet, visible, or infrared. In one embodiment, the light is light with a wavelength of 200–2500 nm. In a further embodiment, the light is light with a wavelength of 600–800 nm, 600–700 nm, or about 630 nm.

[0033] When light-irradiated blood is returned to the target, the cell death cancer cells contained in it are recognized by the target's immune system, and the immune response against the cancer cells in the target is enhanced. Thus, the light is irradiated at a level at which the immune response against cancer cells in the target is enhanced when the light-irradiated blood is collected from the target and returned to the target, that is, at a level at which the light-irradiated blood functions as a vaccine. The level at which the immune response against cancer cells in the target is enhanced is a level at which cancer cells in the target's body other than the light-irradiated cancer cells can be damaged.

[0034] In one embodiment, light is irradiated at a level that increases the proportion of dead cells among cancer cells in blood collected from the subject after the light-irradiated blood is returned to the subject. Dead cells may be apoptotic cells and / or necrotic cells. Cancer cells can be determined by detecting photosensitive substances (e.g., PpIX) accumulated within the cells. Live cells, apoptotic cells, and necrotic cells can be determined by staining with Annexin V and propidium iodide (PI), for example. With staining with Annexin V and PI, Annexin V(-) and PI(-) are determined to be live cells, Annexin V(+) and PI(-) are determined to be apoptotic cells, and Annexin V(+) and PI(+) are determined to be necrotic cells. Staining with Annexin V and PI can be performed using commercially available kits such as the FITC Annexin V Apoptosis Detection Kit I (BD).

[0035] In one embodiment, in photoirradiated cancer cells, the percentage of viable cells immediately after irradiation is 8% or more, and the percentage of viable cells 24 hours after irradiation is 2% or more lower than the percentage of viable cells immediately after irradiation. In a further embodiment, in photoirradiated cancer cells, the percentage of viable cells immediately after irradiation is 8% or more, the percentage of viable cells 24 hours after irradiation is 2% or more lower than the percentage of viable cells immediately after irradiation, and the percentage of apoptotic or necrotic cells 24 hours after irradiation is 2% or more higher than the percentage of the aforementioned cells immediately after irradiation. In this specification, "immediately after irradiation" means within 30 minutes of irradiation.

[0036] For example, in photoirradiated cancer cells, the percentage of viable cells immediately after irradiation may be 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more, and the percentage of viable cells 24 hours after irradiation may be 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more lower than the percentage of viable cells immediately after irradiation. Also, for example, in photoirradiated cancer cells, the percentage of apoptotic cells or necrotic cells 24 hours after irradiation may be 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more higher than the percentage of the aforementioned cells immediately after irradiation.

[0037] In one embodiment, in photoirradiated cancer cells, the percentage of viable cells immediately after irradiation is 20% or more, and the percentage of viable cells 24 hours after irradiation is 10% or more lower than the percentage of viable cells immediately after irradiation. In a further embodiment, in photoirradiated cancer cells, the percentage of viable cells immediately after irradiation is 20% or more, the percentage of viable cells 24 hours after irradiation is 10% or more lower than the percentage of viable cells immediately after irradiation, and the percentage of apoptotic or necrotic cells 24 hours after irradiation is 10% or more higher than the percentage of the aforementioned cells immediately after irradiation.

[0038] When the light-irradiated blood is returned to the target, the percentage of viable cells among the light-irradiated cancer cells may be 8% or more. For example, when the light-irradiated blood is returned to the target, the percentage of viable cells among the light-irradiated cancer cells may be 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more. In one embodiment, when the light-irradiated blood is returned to the target, the percentage of viable cells among the light-irradiated cancer cells is 20% or more.

[0039] In one embodiment, the amount of singlet oxygen generated in the blood collected from the subject by light irradiation is 0.32 × 10⁻¹⁰. -3 Less than mol / L, or 10 9 Irradiation is performed at a level less than one singlet oxygen molecule per cell.

[0040] In certain embodiments, the light is irradiated at an irradiance and for a time such that the irradiation energy density is 0.1 J / cm 2 , 0.5 J / cm 2 , 1 J / cm 2 , 1.5 J / cm 2 , 2 J / cm 2 , 2.5 J / cm 2 , 3 J / cm 2 , 5 J / cm 2 , 7.5 J / cm 2 , 10 J / cm 2 , 20 J / cm 2 , 30 J / cm 2 , 40 J / cm 2 , 50 J / cm 2 , or 100 J / cm 2 or more, 1000 J / cm 2 , 500 J / cm 2 , 400 J / cm 2 , 350 J / cm 2 , 300 J / cm 2 , 250 J / cm 2 , or 200 J / cm 2 or less. In certain embodiments, the irradiation energy density is 1 - 300 J / cm 2 , 2.5 - 300 J / cm 2 , 5 - 300 J / cm 2 , or 10 - 300 J / cm 2 . The irradiation energy density is 10 - 300 J / cm 2 , 50 - 300 J / cm 2 , 100 - 300 J / cm 2 , 100 - 200 J / cm 2 or about 100 J / cm 2 may also be. The irradiation energy density (J / cm 2 ) is obtained by irradiance (W / cm 2 ) × total irradiation time (seconds).

[0041] The irradiance is 1 - 300 mW / cm 2 , 50 - 300 mW / cm 2 , 100 - 300 mW / cm 2 , or 150 - 300 mW / cm 2This is possible. The irradiation may be continuous or intermittent, and the total irradiation time may be, for example, 10 seconds to 30 minutes, 30 seconds to 15 minutes, 1 minute to 10 minutes, or 5 minutes to 10 minutes.

[0042] In one embodiment, the photosensitive substance is 5-ALA, which is administered to the subject at a dose of 10-60 mg / kg or 20-60 mg / kg (e.g., 10, 20, 30, 40, 50, or 60 mg / kg), and 10-300 J / cm³ is added to the blood collected from the subject. 2 Light with a specific irradiation energy density is irradiated.

[0043] The light may be of an irradiation energy density such that the percentage of viable cells 24 hours after irradiation is lower than the percentage of viable cells immediately after irradiation in the test described in Example 3. In one embodiment, the light is used to harvest cancer cells cultured for 4 hours in a medium containing 0.5 mM 5-ALA, suspend them in a red blood cell solution, and then extract 2 × 10⁻⁶ cells. 7This refers to light with an irradiation energy density such that, when applied to a cell suspension adjusted to cells / mL, the percentage of viable cells 24 hours after irradiation is lower than the percentage of viable cells immediately after irradiation. Here, red blood cell solution refers to a solution containing red blood cells and phosphate-buffered saline (PBS) in a 45:55 ratio. Culture is performed at 37°C in a 5% CO2 environment. Any cell culture medium suitable for cancer cells can be used as the culture medium; for example, F-12K medium containing 2.5% FBS or RPMI1640 medium containing 10% FBS can be used. C6 cells or TL-0m1 cells are preferred cancer cells. A wavelength of 630 nm is preferred for the light. Red blood cells are hemolyzed after light irradiation and before measuring the percentage of viable cells (usually within 30 minutes). Hemolysis of red blood cells can be performed by conventional means such as ammonium chloride solution. For example, the light may be light with an irradiation energy density such that the percentage of living cells immediately after irradiation is 8% or more, and the percentage of living cells 24 hours after irradiation is 2% or more lower than the percentage of living cells immediately after irradiation. The light may also be light with an irradiation energy density such that the percentage of living cells immediately after irradiation is 8% or more, the percentage of living cells 24 hours after irradiation is 2% or more lower than the percentage of living cells immediately after irradiation, and the percentage of apoptotic cells or necrotic cells 24 hours after irradiation is 2% or more higher than the percentage of cells immediately after irradiation. The percentage of living cells immediately after irradiation may be 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more, and the percentage of living cells 24 hours after irradiation may be 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more lower than the percentage of living cells immediately after irradiation. Furthermore, the percentage of apoptotic or necrotic cells 24 hours after irradiation may be 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more higher than the percentage of the aforementioned cells immediately after irradiation.

[0044] Light-irradiated blood can enhance the immune response of a target and treat cancer. Therefore, this disclosure includes a method for preparing blood for use in the treatment of cancer.

[0045] A pharmaceutical composition containing a photosensitive substance may also contain a pharmaceutically acceptable carrier and / or additives in addition to the photosensitive substance as the active ingredient. Examples of pharmaceutically acceptable carriers include sterile water, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, lactose, mannitol, crystalline cellulose, hydroxypropyl cellulose, corn starch, and hydroxypropyl methylcellulose. Examples of additives include disintegrants, stabilizers, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, and lubricants. Examples of dosage forms include, but are not limited to, tablets, capsules, powders, granules, liquids, suspensions, injections, and suppositories. The pharmaceutical composition can be formulated by conventional methods.

[0046] The cancer treatments described herein may be used in combination with other cancer drugs. Examples of cancer drugs include vincristine, cyclophosphamide, doxorubicin, prednisolone, ranimustine, vindesine, etoposide, carboplatin, cytarabine, methotrexate, oxaliplatin, cisplatin, dacarbazine, melphalan, ifosfamide, fluorouracil, vinorelbine, idarubicin, epirubicin, gemcitabine, capecitabine, tegafur / gimeracil / oteracil potassium (TS-1), paclitaxel, docetaxel, bevacizumab, erlotinib, gefitinib, trastuzumab, interferon-alpha, tamoxifen, irinotecan, or one or more of these in combination.

[0047] This disclosure provides, for example, the following: [1] It is a system for treating cancer. An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A system that enhances the immune response against cancer cells in the subject by returning blood collected from the subject, irradiated with light, back to the subject. [2] The system according to claim 1, wherein the light is light at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject. [3] It is a system for treating cancer. An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The blood collected from the subject and irradiated with light is returned to the subject. A system in which the light is at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject. [4] The aforementioned light is 2.5~300 J / cm² 2 The system according to any one of the above 1 to 3, wherein the light is of the irradiation energy density. [5] It is a system for treating cancer. An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The blood collected from the subject and irradiated with light is returned to the subject. The aforementioned light is 2.5~300 J / cm² 2 A system of light with a high irradiation energy density. [6] The system according to any one of 3 to 5, wherein the immune response against cancer cells in the subject is enhanced by returning blood collected from the subject and irradiated with light back to the subject. [7] The system according to any one of 1 to 6, wherein the photosensitive substance is administered to the subject prior to blood collection. [8] The system according to any one of 1 to 7, further comprising a photosensitive substance. [9] The system according to any one of claims 1 to 8, wherein the light is light with a wavelength of 200 to 2500 nm.

[10] The system according to 9, wherein the light is light with a wavelength of 600 to 800 nm.

[11] The aforementioned light is 10-300 J / cm² 2 The system according to any one of 1 to 10, wherein the light is of the irradiation energy density.

[12] The aforementioned light is approximately 100 J / cm². 2 The system according to 11, wherein the light is of the irradiation energy density.

[13] The system according to any one of 1 to 12, wherein the photosensitive substance is an ALA compound.

[14] The system according to 13, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[15] The system according to 14, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[16] The system according to 15, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

[17] The system according to 15, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[18] The system according to any one of items 1 to 17, wherein the cancer is a blood cancer.

[19] The system according to 18, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[20] The system according to 19, wherein the blood cancer is ATL. [twenty one] The system according to any one of items 1 to 17, wherein the cancer is a solid tumor. [twenty two] A system according to any one of items 1 to 21 above for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow. [twenty three] A system according to any one of items 1 to 21 above for preventing or treating cancer metastasis. [twenty four] The system according to 23, wherein the cancer metastasis is a lesion in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0048] [twenty five] A method for operating a system for treating cancer, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A method wherein the light irradiation device generates light at a level that enhances the immune response against cancer cells in the target, upon return of blood collected from the target and irradiated with light to the target.

[26] The method according to 25, wherein the light emitted by the light irradiation device is light at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[27] A method for operating a system for treating cancer, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A method wherein the light irradiation device generates light at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[28] The light generated by the aforementioned light irradiation device is 2.5 to 300 J / cm². 2 The method according to any one of the above 25 to 27, wherein the light is of the irradiation energy density.

[29] A method for operating a system for treating cancer, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light irradiation device emits 2.5 to 300 J / cm². 2 A method for generating light with irradiation energy density.

[30] The method according to any one of the 27-29, wherein the light generated by the light irradiation device is at a level that enhances the immune response against cancer cells in the target when blood collected from the target and irradiated with light is returned to the target.

[31] The method according to any one of the 25 to 30, wherein the photosensitive substance is administered to the subject prior to blood collection.

[32] The method according to any one of 25 to 31, wherein the light generated by the light irradiation device is light with a wavelength of 200 to 2500 nm.

[33] The method according to 32, wherein the light generated by the light irradiation device is light with a wavelength of 600 to 800 nm.

[34] The light emitted by the aforementioned light irradiation device is 10-300 J / cm². 2 The method according to any one of the above 25 to 33, wherein the light is of the irradiation energy density.

[35] The light emitted by the aforementioned light irradiation device is approximately 100 J / cm². 2 The method according to 34, wherein the light is of the irradiation energy density.

[36] The method according to any one of the 25 to 35, wherein the photosensitive substance is an ALA compound.

[37] The method according to 36, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[38] The method according to 37, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[39] The method according to 38, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

[40] The method according to 38, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[41] The method according to any one of the above 25 to 40, wherein the cancer is a blood cancer.

[42] The method according to 41, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[43] The method according to 42, wherein the blood cancer is ATL.

[44] The method according to any one of the above 25 to 40, wherein the cancer is a solid tumor.

[45] The method according to any one of the claims 25-44, wherein the system is for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[46] The method according to any one of the above 25 to 44, wherein the system is for preventing or treating cancer metastasis.

[47] The method according to 46, wherein the cancer metastasis is a lesion in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0049]

[48] A pharmaceutical composition comprising a photosensitive substance for treating cancer, used in the following method: (1) Irradiating the blood collected from the subject with light, and (2) Return the light-irradiated blood to the vein of the subject, thereby enhancing the immune response against cancer cells in the subject. Includes, A method comprising administering a photosensitive substance to the subject prior to blood collection, or adding it to blood collected from the subject prior to light irradiation.

[49] The pharmaceutical composition according to 48, wherein the light is light at a level that increases the proportion of dead cells in cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[50] A pharmaceutical composition comprising a photosensitive substance for treating cancer, used in the following method: (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A method wherein the light is at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[51] The aforementioned light is 2.5~300 J / cm² 2 A pharmaceutical composition according to any one of the above 48 to 50, wherein the light is of the irradiation energy density.

[52] A pharmaceutical composition comprising a photosensitive substance for treating cancer, used in the following method: (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light is 2.5~300 J / cm² 2 A method in which the irradiation energy density of light is high.

[53] The pharmaceutical composition according to any one of 50 to 52, wherein the method enhances the immune response against cancer cells in the target by returning the light-irradiated blood to the target.

[54] The pharmaceutical composition according to any one of the 48 to 53, wherein the method further comprises collecting blood from the subject prior to step (1)'.

[55] The pharmaceutical composition according to 54, wherein the method further comprises administering a photosensitive substance to the subject prior to step (1)''.

[56] The pharmaceutical composition according to 54, wherein the method further comprises adding a photosensitive substance to the blood collected from the subject after step (1) and prior to step (1).

[57] A pharmaceutical composition according to any one of 48 to 56, wherein blood collection and return are performed by an extracorporeal circulation device.

[58] The pharmaceutical composition according to any one of the 48 to 57, wherein the light is light with a wavelength of 200 to 2500 nm.

[59] The pharmaceutical composition according to 58, wherein the light is light with a wavelength of 600 to 800 nm.

[60] The aforementioned light is 10-300 J / cm² 2 A pharmaceutical composition according to any one of the above 48 to 59, wherein the light is of the irradiation energy density.

[61] The aforementioned light is approximately 100 J / cm². 2 The pharmaceutical composition according to 60, wherein the light is light with an irradiation energy density.

[62] The pharmaceutical composition according to any one of the above 48 to 61, wherein the photosensitive substance is an ALA compound.

[63] The pharmaceutical composition according to 62, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[64] The pharmaceutical composition according to 63, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[65] The pharmaceutical composition according to 64, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

[66] The pharmaceutical composition according to 64, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[67] The pharmaceutical composition according to any one of the above 48 to 66, wherein the cancer is a blood cancer.

[68] The pharmaceutical composition according to 67, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[69] The pharmaceutical composition according to 68, wherein the blood cancer is ATL.

[70] The pharmaceutical composition according to any one of the above 48 to 66, wherein the cancer is a solid tumor.

[71] A pharmaceutical composition according to any one of the above 48 to 70 for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[72] A pharmaceutical composition according to any one of the above 48 to 70 for preventing or treating cancer metastasis.

[73] The pharmaceutical composition according to 72, wherein the cancer metastasis is a lesion of the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0050]

[74] A method of treating cancer, (1) Irradiating the blood collected from the subject with light, and (2) Return the light-irradiated blood to the vein of the subject, thereby enhancing the immune response against cancer cells in the subject. Includes, A method comprising administering a photosensitive substance to the subject prior to blood collection, or adding it to blood collected from the subject prior to light irradiation.

[75] The method according to 74, wherein the light is light at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[76] A method of treating cancer, (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A method wherein the light is at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[77] The aforementioned light is 2.5~300 J / cm² 2 The method according to any one of 74 to 76, wherein the light is of the irradiation energy density.

[78] A method of treating cancer, (1) Irradiating the blood collected from the subject with light, and (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light is 2.5~300 J / cm² 2 A method in which the irradiation energy density of light is high.

[79] The method according to any one of the 76 to 78, wherein the light-irradiated blood is returned to the target to enhance the immune response against cancer cells in the target.

[80] (1)' The method according to any one of 74 to 79, further comprising taking blood from the subject prior to step (1).

[81] (1) The method according to 80, further comprising administering a photosensitive substance to the subject prior to step (1)'.

[82] (1) The method according to 80, further comprising adding a photosensitive substance to the blood collected from the subject after step (1) and prior to step (1).

[83] The method according to any one of items 74 to 82, wherein blood collection and return are performed by an extracorporeal circulation device.

[84] The method according to any one of the above 74 to 83, wherein the light is light with a wavelength of 200 to 2500 nm.

[85] The method according to 84, wherein the light is light with a wavelength of 600 to 800 nm.

[86] The aforementioned light is 10-300 J / cm² 2 The method according to any one of the above 74 to 85, wherein the light is of the irradiation energy density.

[87] The aforementioned light is approximately 100 J / cm². 2 The method according to 86, wherein the light is of the irradiation energy density.

[88] The method according to any one of the above 74 to 87, wherein the photosensitive substance is an ALA compound.

[89] The method according to 88, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[90] The method according to 89, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[91] The method according to 90, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

[92] The method according to 90, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[93] The method according to any one of items 74 to 92, wherein the cancer is a blood cancer.

[94] The method according to 93, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[95] The method according to 94, wherein the blood cancer is ATL.

[96] The method according to any one of items 74 to 92, wherein the cancer is a solid tumor.

[97] The method according to any one of the preceding 74-96 for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[98] A method according to any one of the above 74 to 96 for preventing or treating cancer metastasis.

[99] The method according to 98, wherein the cancer metastasis is a lesion in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0051]

[0100] A system for preventing or treating cancer metastasis, An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. A system for preventing the development of metastatic lesions or treating metastatic lesions by returning blood collected from the subject, irradiated with light, back to the subject.

[0101] The system according to 100, wherein the light is light at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[0102] A system for preventing or treating cancer metastasis, An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The blood collected from the subject and irradiated with light is returned to the subject. A system in which the light is at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[0103] The aforementioned light is 2.5~300 J / cm² 2 The system according to any one of the above 100 to 102, wherein the light is of the irradiation energy density.

[0104] A system for preventing or treating cancer metastasis, An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The blood collected from the subject and irradiated with light is returned to the subject. The aforementioned light is 2.5~300 J / cm² 2 A system of light with a high irradiation energy density.

[0105] The system according to any one of 100 to 104, wherein the immune response against cancer cells in the subject is enhanced by returning blood collected from the subject and irradiated with light back to the subject.

[0106] The system according to any one of claims 100 to 105, wherein the photosensitive substance is administered to the subject prior to blood collection.

[0107] The system according to any one of 100 to 106, further comprising a photosensitive substance.

[0108] The system according to any one of claims 100 to 107, wherein the light is light with a wavelength of 200 to 2500 nm.

[0109] The system according to 108, wherein the light is light with a wavelength of 600 to 800 nm.

[0110] The aforementioned light is 10-300 J / cm² 2 The system according to any one of the above 100 to 109, wherein the light is of the irradiation energy density.

[0111] The aforementioned light is approximately 100 J / cm². 2 The system according to 110, wherein the light is of the irradiation energy density.

[0112] The system according to any one of the above 100 to 111, wherein the photosensitive substance is an ALA compound.

[0113] The system according to 112, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[0114] The system according to 113, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[0115] The system according to 114, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

[0116] The system according to 114, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[0117] The system according to any one of items 100 to 116, wherein the cancer is a blood cancer.

[0118] The system according to 117, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[0119] The system according to 118, wherein the blood cancer is ATL.

[0120] The system according to any one of items 100 to 116, wherein the cancer is a solid tumor.

[0121] The system according to any one of the 100 to 120, wherein the cancer metastasis is a lesion of the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0052]

[0122] A method for preparing blood for use in cancer treatment, Adding a photosensitive substance to blood collected from the subject, and, Irradiating the blood to which a photosensitive substance has been added with light. A method comprising administering the light-irradiated blood into a target vein.

[0123] The method according to 122, wherein the light-irradiated blood enhances the immune response against cancer cells in the subject.

[0124] The method according to 122 or 123, wherein the light is light with a wavelength of 200 to 2500 nm.

[0125] The method according to 124, wherein the light is light with a wavelength of 600 to 800 nm.

[0126] The method according to any one of 122 to 125, wherein the light is light at a level that increases the proportion of dead cells in cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[0127] The aforementioned light is 2.5~300 J / cm² 2 The method according to any one of 122 to 126, wherein the light is of the irradiation energy density.

[0128] The aforementioned light is 10-300 J / cm² 2 The method according to any one of the above 122 to 127, wherein the light is of the irradiation energy density.

[0129] The aforementioned light is approximately 100 J / cm². 2 The method according to 128, wherein the light is of the irradiation energy density.

[0130] The method according to any one of 122 to 129, wherein the photosensitive substance is an ALA compound.

[0131] The method according to 130, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[0132] The method according to 131, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[0133] The method according to 132, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[0134] The method according to any one of items 122 to 133, wherein the cancer is a blood cancer.

[0135] The method according to 134, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[0136] The method according to 135, wherein the blood cancer is ATL.

[0137] The method according to any one of the above 122 to 133, wherein the cancer is a solid tumor.

[0138] The method according to any one of the claims 122 to 137, wherein the blood is for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0139] The method according to any one of items 122 to 137, wherein the blood is intended for use in the prevention or treatment of cancer metastasis.

[0140] The method according to 139, wherein the cancer metastasis is a lesion in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0053]

[0141] A method for preparing blood for use in the prevention or treatment of cancer metastasis, Adding a photosensitive substance to blood collected from the subject, and, Irradiating the blood to which a photosensitive substance has been added with light. A method comprising administering the light-irradiated blood into a target vein.

[0142] The method according to 141, wherein the light-irradiated blood enhances the immune response against cancer cells in the subject.

[0143] The method according to 141 or 142, wherein the light is light with a wavelength of 200 to 2500 nm.

[0144] The method according to 143, wherein the light is light with a wavelength of 600 to 800 nm.

[0145] The method according to any one of 141 to 144, wherein the light is at a level that increases the proportion of dead cells in the cancer cells contained in the blood collected from the subject after the blood is returned to the subject.

[0146] The aforementioned light is 2.5~300 J / cm² 2 The method according to any one of the above 141 to 145, wherein the light is of the irradiation energy density.

[0147] The aforementioned light is 10-300 J / cm² 2 The method according to any one of the above 1141 to 146, wherein the light is of the irradiation energy density.

[0148] The aforementioned light is approximately 100 J / cm². 2 The method according to 147, wherein the light is of the irradiation energy density.

[0149] The method according to any one of the above 141 to 148, wherein the photosensitive substance is an ALA compound.

[0150] The method according to 149, wherein the ALA compound is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

[0151] The method according to 150, wherein the ALA compound is 5-ALA or a pharmaceutically acceptable salt thereof.

[0152] The method according to 151, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

[0153] The method according to any one of items 141 to 152, wherein the cancer is a blood cancer.

[0154] The method according to 153, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

[0155] The method according to 154, wherein the blood cancer is ATL.

[0156] The method according to any one of items 141 to 152, wherein the cancer is a solid tumor.

[0157] The method according to any one of items 141 to 156, wherein the cancer metastasis is a lesion in the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

[0054]

[0158] A photosensitive substance for use in the treatment of cancer, which is used in the method described in any of the above 74 to 99.

[0055]

[0159] Use of a photosensitive substance for the manufacture of a pharmaceutical for use in the treatment of cancer, wherein the pharmaceutical is used in any of the methods described in 74 to 99 above.

[0056]

[0160] A method for operating a system for treating cancer, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light irradiation device collects cancer cells cultured for 4 hours in a medium containing 0.5 mM 5-ALA, and suspends them in red blood cell solution for 2 × 10⁶ cells. 7A method for generating light with an irradiation energy density such that, when irradiated onto a cell suspension adjusted to cells / mL, the percentage of viable cells 24 hours after irradiation is lower than the percentage of viable cells immediately after irradiation.

[0161] The method according to 160, wherein the light generated by the light irradiation device is light with an irradiation energy density such that, when irradiated onto the cell suspension, the percentage of living cells immediately after irradiation is 8% or more, and the percentage of living cells 24 hours after irradiation is 2% or more lower than the percentage of living cells immediately after irradiation.

[0162] The method according to 160 or 161, wherein the light generated by the light irradiation device is light with an irradiation energy density such that, when irradiated onto the cell suspension, the percentage of living cells immediately after irradiation is 8% or more, the percentage of living cells 24 hours after irradiation is 2% or more lower than the percentage of living cells immediately after irradiation, and the percentage of apoptotic cells or necrotic cells 24 hours after irradiation is 2% or more higher than the percentage of cells immediately after irradiation.

[0163] The method according to any one of items 160 to 162, wherein the light generated by the light irradiation device is light with a wavelength of 630 nm.

[0057] The present invention will be further illustrated by the following embodiments, but in no sense is the present invention limited to these embodiments. [Examples]

[0058] Example 1 1. Method Intravenous administration of PDT-treated C6 cells Rat glioma cell line C6 cells (ATCC) were cultured for 24 hours in a 37°C 5% CO2 incubator in 2.5% FBS / F-12K medium (Thermo Fisher Scientific) supplemented with aminolevulinic acid (5-ALA) (Sigma-Aldrich) to a final concentration of 1 mM. After culturing, the cells were washed with PBS and treated with 0.25% trypsin-EDTA solution (Thermo Fisher Scientific), followed by 2 × 10⁶ solutions in PBS.7 The cell suspension was adjusted to cells / mL. The adjusted cell suspension was injected into a Tygon tube (inner diameter: 0.79 mm, Saint-Gobain), wrapped around a light curing unit (Otsuka Electronics), and irradiated with 630 nm light. Irradiation was performed under the following two irradiation conditions (PDT-1 and PDT-2). PDT-1 Illuminance: 264.2mW / cm 2 Time: 20 minutes Irradiation energy density: 317.0 J / cm² 2 PDT-2 Illuminance: 124.5mW / cm 2 (First time) 91.4 mW / cm² 2 (2nd and 3rd time) * ) Time: 1 minute Irradiation energy density: 7.5 J / cm² 2 (First time) 5.5J / cm 2 (2nd and 3rd time) * ) *Based on the first cell death rate, the illumination was adjusted to a lower level. Treatment cells were pooled according to each irradiation condition, and irradiation was carried out until the required dose was obtained. Under isoflurane (Pfizer) anesthesia, 0.5 mL of each PDT-treated cell was administered to each F344 rat (8 weeks old at the start of the study, male, Charles River, Japan) (1 × 10⁶). 7 A number of cells equivalent to the total number of cells were administered via tail vein (3 animals in each group). As a control, 0.5 mL of PBS alone was administered intravenously to 3 animals. A total of three intravenous administrations of these PDT-treated cells were performed (Day 21, Day 14, Day 1).

[0059] PDT-treated C6 cell death rate PDT-treated C6 cells were stained with FITC-labeled Annexin V and PI using the FITC Annexin V Apoptosis Detection Kit I (BD). The cell death rate was then analyzed using a flow cytometer (FACSCalibur, BD).

[0060] Subcutaneous transplantation of C6 cells (creation of tumor-bearing rats) On the day following the third intravenous administration of PDT-treated cells (Day 0), C6 cells washed with PBS were treated with 0.25% trypsin-EDTA solution and then 2 × 10⁶ cells were hydrated in PBS. 7 The solution was adjusted to cells / mL. Under isoflurane anesthesia, 0.5 mL (1 × 10⁶) was administered per F344 rat. 7 Cells were subcutaneously transplanted into the left flank.

[0061] Calculation of tumor volume On days 3, 6, 8, and 10 after tumor-bearing rats were created, the longest and shortest diameters of the tumors were measured using calipers, and the tumor volume was calculated using the formula shown below. Tumor volume = 1 / 6 × π × major diameter × minor diameter 2

[0062] Tumor-infiltrated CD8 + Measurement of T cells On day 10, euthanasia was performed by total blood collection under isoflurane anesthesia, and the tumor was removed. The removed tumor was fixed with formalin (Wako Pure Chemical Industries), and tissue sections were prepared and immunohistochemical staining (CD8) was performed. + T cell staining was performed. After staining, three fields of view were randomly selected at 400x magnification for CD8 + T cells were measured.

[0063] IFN-γ production by spleen cells The spleen was also removed from the rats that had been euthanized, minced finely, and handled with gauze to squeeze out spleen cells. After washing the collected spleen cells, they were separated into monocytes using a density gradient separation solution (Lympholyte-Rat, Cedarlane). After washing again, they were adjusted to 1×10 6 cells / mL with 10% FBS / RPMI1640 medium. Also, C6 cells were washed with PBS, treated with 0.25% trypsin-EDTA solution, and adjusted to 1×10 5 cells / mL. Then, 100 μL each of spleen cells and C6 cells were added to a 96-well flat-bottom plate (Sumitomo Bakelite) and cultured in a 5% CO2 incubator at 37°C for 24 hours. The culture supernatant after culture was collected, and the amount of IFN-γ was measured by ELISA (R&D Systems).

[0064] 2. Results The protocol of this experiment is shown in Figure 2. Almost 100% of the PDT-1-treated cells were Annexin V(+) and PI(+) immediately after irradiation, and they were necrotic cells (Figure 2). The PDT-2-treated cells contained about 20% viable cells that were Annexin V(-) and PI(-) immediately after irradiation, and among the dead cells, apoptotic cells that were Annexin V(+) and PI(-) and necrotic cells that were Annexin V(+) and PI(+) were mixed, and the cells that were Annexin V(+) and PI(-) were about 5 - 20%, but almost all cells became Annexin V(+)PI(+) 24 hours after irradiation. Figure 3 shows the changes in tumor volume after subcutaneous transplantation of C6 cells in rats that were administered PBS, PDT-1-treated cells, or PDT-2-treated cells before subcutaneous transplantation of C6 cells. In rats that were administered PBS before subcutaneous transplantation of C6 cells, an increase in tumor volume was observed after subcutaneous transplantation of C6 cells. On the other hand, in rats that were administered PDT-2-treated cells, this increase in tumor volume was suppressed. No such suppression was observed with the administration of PDT-1-treated cells.

[0065] On the 10th day (Day10) after subcutaneous transplantation of C6 cells, tumor infiltrating CD8 +IFN-γ production by T cells and spleen cells was higher in rats administered with PDT-2-treated cells than in rats administered with PBS and PDT-1-treated cells (Figure 4).

[0066] The above results indicate that immunity against C6 cells was activated and the growth of transplanted C6 cells was suppressed in rats administered with PDT-2-treated cells.

[0067] Example 2 1. Method Intravenous administration of PDT-treated C6 cells C6 cells were cultured in 2.5% FBS / F-12K medium supplemented with 5-ALA to a final concentration of 1 mM in a 5% CO2 incubator at 37 °C for 24 hours. After culturing, the cells were detached with a 0.25% trypsin-EDTA solution, and the collected cells were adjusted to 2×10 7 cells / mL with PBS. The adjusted cell suspension was injected into a Tygon tube (inner diameter: 0.79 mm), wrapped around a light irradiator (Otsuka Electronics), and irradiated with light at 630 nm with an irradiation energy density of 7.5 J / cm 2 (an illuminance of 124.5 mW / cm 2 for 1 minute). The treated cells were pooled and irradiated until the required dose was obtained. The irradiated cells were divided into three equal parts. One part was administered via the tail vein to each of three F344 rats (8 weeks old, male, Charles River Japan) at 0.5 mL (1×10 7 cells) per rat under isoflurane anesthesia (PDT iv group). Another part was adjusted to 2×(10 7 cells / mL with PBS after centrifugation and removal of the supernatant, and administered via the tail vein to each rat at 0.5 mL (1×10 7 cells) per rat in the same manner as the PDT iv group (PDT-cell iv group, 3 rats). The remaining one-third of the divided cells were suspended in 2.5% FBS / F-12K medium, cultured in a 5% CO2 incubator at 37 °C for 24 hours, and the collected cells were adjusted to 2×10 7 cells / mL with PBS and administered via the tail vein to each rat at 0.5 mL (1×10 7Cells were administered via tail vein one at a time (PDT / 24h-cell iv group, 3 animals). A total of three intravenous administrations of these PDT-treated cells were performed (Day 15, Day 8, Day 1).

[0068] Intravenous administration of C6 cells treated with anticancer drugs C6 cells were cultured for 48 hours in a 5% CO2 incubator at 37°C in 2.5% FBS / F-12K medium to which mitoxantrone dihydrochloride (MTX) (Sigma-Aldrich), doxorubicin hydrochloride (DOX) (Sigma-Aldrich), and epirubicin hydrochloride (EPI) (Sigma-Aldrich) were added to final concentrations of 20 μg / mL, 75 μg / mL, and 62.5 μg / mL, respectively. After culturing, the cells were detached with 0.25% trypsin-EDTA solution, and the collected cells were refrigerated in PBS in 2 × 10⁶ cells. 7 The solution was adjusted to cells / mL. The anticancer drug-treated cells were administered to each F344 rat in 0.5 mL (1 × 10⁶) under isoflurane anesthesia. 7 Cells were administered via tail vein in groups of three (MTX-cell iv, DOX-cell iv, and EPI-cell iv). A total of three intravenous administrations of these treated cells were performed (Day 15, Day 8, Day 1).

[0069] Death rate of C6 cells treated with PDT or anticancer drugs Using the FITC Annexin V Apoptosis Detection Kit I, C6 cells treated with PDT or anticancer drugs were stained with FITC-labeled Annexin V and PI. The cell death rate was then analyzed using a flow cytometer.

[0070] Subcutaneous transplantation of C6 cells (creation of tumor-bearing rats) On the day following the third intravenous administration of PDT-treated or anticancer drug-treated cells (Day 0), untreated C6 cells were detached with 0.25% trypsin-EDTA solution, collected, and then stored in PBS in 2 × 10⁶ cells. 7 The cells were adjusted to a concentration of cells / mL. These cells were then administered to 0.5 mL (1 × 10⁶) per F344 rat under isoflurane anesthesia. 7They were subcutaneously transplanted into the left abdomen (cells).

[0071] Calculation of tumor volume On the 4th, 6th, 8th, and 10th days (Day 4, 6, 8, 10) after the establishment of cancer-bearing rats, the major and minor diameters of the tumors were measured using calipers, and the tumor volume was calculated using the following formula. Tumor volume = 1 / 6 × π × major diameter × minor diameter 2

[0072] Measurement of DAMPs (Damage-associated molecular patterns) C6 cells treated with PDT or anticancer agents were stained with FITC-labeled anti-Calreticulin antibody (Bioss) and FITC-labeled isotype control (Bioss), and the fluorescence intensity of Calreticulin was analyzed using a flow cytometer. In addition, the HMGB1 concentration in the supernatant obtained by centrifuging C6 cells treated with PDT or anticancer agents was measured using HMGB1 ELISA Kit II (Sinotest).

[0073] 2. Results The changes in tumor volume after subcutaneous transplantation of C6 cells in the PDT iv group, PDT-cell iv group, PDT / 24h-cell iv group, MTX-cell iv group, DOX-cell iv group, and EPI-cell iv group, and the group administered PBS (0.5 mL) intravenously as a control (PBS iv group) are shown in Fig. 5, and the tumor tissues excised on the 10th day after subcutaneous transplantation of C6 cells are shown in Fig. 6. An increase in tumor volume was observed in the PBS iv group, whereas this increase in tumor volume was suppressed in the PDT iv group, PDT-cell iv group, and PDT / 24h-cell iv group. On the other hand, this suppression was not observed in the MTX-cell iv group, DOX-cell iv group, and EPI-cell iv group.

[0074] Anticancer drug-treated cells contained very few viable cells (Annexin V- / PI-), with MTX-treated cells containing 1%, DOX-treated cells 5%, and EPI-treated cells 5%. On the other hand, PDT-treated cells contained approximately 20-60% viable cells immediately after treatment and after 24 hours of culture. In PDT-treated cells, immediately after irradiation, apoptotic cells (Annexin V+ / PI-) and necrotic cells (Annexin V+ / PI+) were mixed among the dead cells. It was revealed that the proportion of viable cells decreased while the proportion of apoptotic and necrotic cells increased 24 hours after treatment, indicating that viable cells gradually died.

[0075] Calreticulin expression and HMGB1 release in PDT-treated cells were not high immediately after PDT treatment, but increased 24 hours after treatment. In anticancer drug-treated cells, both calreticulin expression and HMGB1 release were strongly induced.

[0076] The results are summarized in Table 1. Intravenous administration of PDT-treated cells was confirmed to induce antitumor immunity. This effect was observed with cells immediately after PDT treatment, cells cultured for 24 hours, and even when only the cells were administered after removing the culture supernatant. It is thought that the gradual death of PDT-treated cells triggers the expression and release of DAMPs in vivo, thereby inducing antitumor immunity. On the other hand, while MTX, DOX, and EPI have been reported to have immunostimulatory effects when administered subcutaneously, intravenous administration of cells treated with these substances did not induce antitumor immunity. [Table 1] * HMGB1 release was confirmed by PDT treatment and anticancer drug treatment, but since only treated cells with the supernatant removed were administered intravenously, the results are enclosed in parentheses. In addition, the "+" in the antitumor immunity column indicates that the increase in tumor volume was suppressed.

[0077] Example 3 1. Method C6 cells or the human leukemia cell line TL-Om1 cells were cultured for 4 hours in a 37°C 5% CO2 incubator in 2.5% FBS / F-12K medium (C6 cells) or 10% FBS / RPMI1640 medium (TL-Om1 cells) supplemented with 5-ALA to a final concentration of 0.125-0.5 mM. After culturing, the cells were detached with 0.25% trypsin-EDTA solution, and the collected cells were fermented in erythrocyte solution (a solution of erythrocytes and PBS mixed in a 45:55 ratio) for 2 × 10⁶ times. 7 The cell suspension was adjusted to a concentration of cells / mL. The adjusted cell suspension was injected into a Tygon tube (inner diameter: 0.79 mm), wrapped around a light curing unit, and irradiated with 630 nm light at an energy density of 0.5-500 J / cm². 2 The cells were irradiated to achieve the desired result. After irradiation, ammonium chloride solution was added to the cell suspension to lyse red blood cells. Cells were stained using the FITC Annexin V Apoptosis Detection Kit I before, after, and 24 hours after PDT treatment, and the rate of dead cells was analyzed using a flow cytometer.

[0078] 2.Results The results are summarized in Tables 2 and 3. [Table 2] [Table 3]

[0079] Based on the results in Tables 2 and 3, we investigated the conditions under which PDT-treated cells gradually die. We focused on the results for final concentrations of 0.25 mM and 0.5 mM, which were considered to have added a sufficient amount of 5-ALA to the cells based on the changes in irradiation energy density and the percentage of viable cells. Considering the results of Examples 1 and 2, we investigated the irradiation energy density at which the percentage of viable cells immediately after irradiation was 8% or more, and the percentage of viable cells 24 hours after irradiation was 2% or more lower than the percentage of viable cells immediately after irradiation. The results for C6 cells and TL-0m1 cells, respectively, were as follows. Under these conditions, the percentage of apoptotic or necrotic cells 24 hours after irradiation was 2% or more higher than the percentage of the aforementioned cells immediately after irradiation. [Table 4] From the results of Examples 1-3, the irradiation energy density was 2.5-300 J / cm². 2 This is preferable, and especially 10-300 J / cm². 2 This demonstrated that the method is applicable regardless of the cell type.

Claims

1. A method for operating a system for preventing or treating cancer metastasis, The aforementioned system An extracorporeal circulation device, including a blood circuit and blood pump, for collecting and returning the target blood, and A light irradiation device for irradiating the blood circuit with light. Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light irradiation device has a light intensity of 30 to 300 J / cm². ２ It generates light with an irradiation energy density of, A method wherein the photosensitive substance is 5-aminolevulinic acid (5-ALA), its methyl ester, ethyl ester, propyl ester, hexyl ester, heptyl ester, or octyl ester, or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein the light generated by the light irradiation device is at a level that enhances the immune response against cancer cells in the target when blood collected from the target and irradiated with light is returned to the target.

3. The method according to claim 1 or 2, wherein the system is for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

4. The method according to any one of claims 1 to 3, wherein the cancer metastasis is a lesion of the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

5. The method according to any one of claims 1 to 4, wherein the light generated by the light irradiation device is light with a wavelength of 200 to 2500 nm.

6. The method according to any one of claims 1 to 5, wherein the photosensitive substance is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

7. The method according to claim 6, wherein the photosensitive substance is 5-ALA or a pharmaceutically acceptable salt thereof.

8. The method according to claim 7, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

9. The method according to claim 7, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

10. The method according to any one of claims 1 to 9, wherein the cancer is a blood cancer.

11. The method according to claim 10, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

12. The method according to claim 11, wherein the blood cancer is ATL.

13. A pharmaceutical composition comprising a photosensitive substance for preventing or treating cancer metastasis, used in the following methods: (1) Irradiating the blood collected from the subject with light, (2) Returning the light-irradiated blood to the target vein, Includes, A photosensitive substance is administered to the subject prior to blood collection, or added to the blood collected from the subject prior to light irradiation. The aforementioned light is 30-300 J / cm². ２ It is light with an irradiation energy density, A method wherein the photosensitive substance is 5-aminolevulinic acid (5-ALA), its methyl ester, ethyl ester, propyl ester, hexyl ester, heptyl ester, or octyl ester, or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical composition according to claim 13, wherein the method enhances the immune response against cancer cells in the target by returning the light-irradiated blood to the target.

15. A pharmaceutical composition according to claim 13 or 14 for treating lesions of peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

16. The pharmaceutical composition according to any one of claims 13 to 15, wherein the cancer metastasis is a lesion of the peripheral blood, lymph nodes, spleen, liver or other extranodal organs, skin, or bone marrow.

17. The pharmaceutical composition according to any one of claims 13 to 16, wherein the photosensitive substance is 5-aminolevulinic acid (5-ALA), its methyl ester or hexyl ester, or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition according to claim 17, wherein the photosensitive substance is 5-ALA or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to claim 18, wherein 5-ALA or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 1 mg to 100 mg / kg.

20. The pharmaceutical composition according to claim 18, wherein 5-ALA or a pharmaceutically acceptable salt thereof is added to blood collected from the subject at a concentration of 0.01 mmol to 10 mmol / L.

21. The pharmaceutical composition according to any one of claims 13 to 20, wherein the cancer is a blood cancer.

22. The pharmaceutical composition according to claim 21, wherein the blood cancer is adult T-cell leukemia (ATL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL).

23. The pharmaceutical composition according to claim 22, wherein the blood cancer is ATL.

24. A method for preparing blood for use in the prevention or treatment of cancer metastasis, Adding a photosensitive substance to blood collected from the subject, and, Irradiating the blood to which a photosensitive substance has been added with light. The blood, which has been irradiated with light, is administered to the target vein, and the light intensity is 30 to 300 J / cm². ２ It is light with an irradiation energy density, A method wherein the photosensitive substance is 5-aminolevulinic acid (5-ALA), its methyl ester, ethyl ester, propyl ester, hexyl ester, heptyl ester, or octyl ester, or a pharmaceutically acceptable salt thereof.

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