Cancer contrast-enhancing composition

Abstract

The present invention addresses the problem of providing a contrast imaging agent by which a cancer tissue can be specifically identified.　The administration of a contrast imaging composition for cancer, said composition containing a compound according to the present invention, enables contrast imaging of a cancer tissue using near-infrared light.

Description

[Technical Field]

[0001] This invention relates to cancer contrast agents. In particular, this invention relates to cancer contrast compositions containing cyclodextrin-bound indocyanine compounds. [Background technology]

[0002] Cancer is the leading cause of death in Japan, and globally, the number of deaths from cancer is increasing, consistently ranking among the top causes of death. While surgical treatment for cancer has long been widely used as a treatment method that offers the possibility of a complete cure, the inability to accurately identify the location of the tumor and the resulting recurrence and worsening due to incomplete removal remain major medical problems. Furthermore, in recent years, improvements in surgical precision have led to an increase in the choice of partial resection, but the risk of incomplete removal always exists, and the expectation for accurate tumor location identification is growing.

[0003] Non-invasive techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) imaging are used for preoperative tumor location identification in cancer surgery, playing an important role in preoperative diagnosis and surgical procedure planning. However, these techniques do not provide actual tumor location information during surgery, and confirmation of the tumor location during surgery relies solely on the surgeon's visual inspection. Therefore, they do not help to improve resection failure caused by bleeding or obstruction of visibility by adjacent tissue.

[0004] Intraoperative guided diagnosis using fluorescence imaging is a technique that is increasingly being used to support visual identification of tumor locations. For example, in cystoscopy for bladder cancer, macroscopic diagnosis under white light and observation of red fluorescence by irradiating protoporphyrin IX, which is produced by the administration of 5-aminolevulinic acid (ALA) or hexyl-5-aminolevulinic acid ester (H-ALA), with blue light (photodynamic diagnosis) are performed. These diagnoses are used to determine the location of bladder cancer tissue, but photodynamic diagnosis with ALA or H-ALA administration makes it easier to confirm cancer tissue than macroscopic diagnosis under white light, reducing the risk of residual cancer tissue during resection and thus reducing cancer recurrence. Currently, H-ALA is sold by Photocure (Norway, https: / / www.photocure.com / ) under brand names such as Hexvix (Europe) and Cysview (USA and Canada) (Non-patent Literature 1). Furthermore, methods have been reported, such as dendrimerization of H-ALA (Non-Patent Literature 2) and nanoparticleization (Non-Patent Literature 3), with the aim of improving the diagnostic capabilities of photodynamic diagnosis using ALA or H-ALA administration.

[0005] Furthermore, the distinction between normal and damaged areas of kidney tissue is made using techniques such as CT image analysis and pathological examination involving excision, staining, and microscopic observation. Intraoperative identification of damaged areas of kidney tissue is also performed by visual inspection.

[0006] Furthermore, recent technological advancements have led to the widespread adoption of fluorescence imaging techniques using near-infrared wavelengths, which easily penetrate biological components, and their medical applications are beginning to spread in various fields. Although near-infrared wavelengths are undetectable to the human eye, they can be detected through CCD cameras and other devices, making them an effective tool for assisting visualization during surgery.

[0007] Indocyanine green (ICG) is a known fluorescent reagent that utilizes near-infrared wavelengths and is marketed in Japan as a drug for liver and circulatory function tests, a fluorescent angiography contrast agent, and a sentinel lymph node identification agent. The sentinel lymph node is the first lymph node that cancer cells reach after entering the lymphatic vessels from the primary tumor, and it is the lymph node with the highest probability of metastasis. The procedure of identifying and removing this lymph node and determining the presence or absence of cancer metastasis through pathological diagnosis is called sentinel lymph node biopsy (SNB). Although ICG is used for sentinel lymph node identification, it does not have a tumor-specific contrast enhancement effect due to its high protein binding ability, and it is not easy to image the tumor itself using near-infrared fluorescence technology (Non-Patent Literature 4).

[0008] Although drug development aimed at tumor fluorescence enhancement is currently underway worldwide, no such drugs are yet available for use in clinical practice.

[0009] Patent Document 1 describes a specific cyclodextrin-bound indocyanine compound as a fluorescent reagent utilizing near-infrared wavelengths, and states that a diagnostic composition containing this compound can be applied to conventional visualization procedures such as fundus angiography, cerebral circulation evaluation, intraoperative angiography in neurosurgery, sentinel lymph node identification in cancer (breast cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, skin cancer, etc.), lymphedema evaluation, intraoperative cholangiography, tumor marking, coronary angiography, and abdominal angiography (hepatic artery, abdominal aorta, gastrointestinal blood flow, etc.). [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent Publication No. 2012 / 0302881 [Non-patent literature]

[0011] [Non-Patent Document 1] Michael Rink et al., European Urology, 2013, 64, 624-638. [Non-Patent Document 2] Francois, Aurelie et al., BJU International, 2012, 110(11c), E1155-1162. [Non-Patent Document 3] Heuck Gesine et al., Journal of Porphyrins and Phthalocyanines, 2012, 16(7-8), 878-884. [Non-Patent Document 4] Shenglin Luo, Erlong Zhang, Yongping Su, Tianmin Cheng, Chunmeng Shi. A review of NIR dyes in cancer targeting and imaging, Biomaterials, 2011, 32 (29), 7127-7138. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] Photodynamic diagnostics using ALA or H-ALA involve detecting the fluorescence of red light after irradiation with blue light. However, this causes the entire bladder to appear blue or bluish-purple, and cancerous tissue is visually observed in red against this background, making it difficult to diagnose deep tissues or to determine the boundary between normal and cancerous tissue (A. Kolodziej, W. Krajewski, M. Matuszewski, K. Tupikowski. Review of current optical diagnostic techniques for non-muscle-invasive bladder cancer, Cent European J Urol. 2016; 69: 150-156). Furthermore, blue light has poor penetration into biological tissue, which may prevent the detection of tumors embedded in surrounding tissues.

[0013] Furthermore, while the identification (contrast enhancement) of sentinel lymph nodes using ICG and other methods is useful because it provides structural information about the lymphatic vessel-lymph node connection to the tumor, it does not allow observation of the tumor itself.

[0014] In general, imaging or identifying tumors themselves using near-infrared fluorescent reagents remains difficult even with recent technological advancements. Although drug development aimed at tumor fluorescence enhancement is being conducted worldwide, no such drugs are yet available for use in clinical practice.

[0015] In light of these circumstances, the object of the present invention is to develop a contrast agent that can specifically identify or distinguish cancerous tissue. [Means for solving the problem]

[0016] The inventors of the present invention conducted extensive research to solve the above problems and found that by using a compound in which cyclodextrin is bound to an indocyanine compound, cancerous tissue can be specifically identified or distinguished by irradiation with near-infrared light. Specifically, the inventors administered a compound indocyanine compound bound to cyclodextrin and irradiated it with near-infrared light. They confirmed that normal tissue did not emit near-infrared fluorescence from this compound, while cancerous tissue did. Furthermore, they confirmed that cancerous tissue in certain types of cancer emitted near-infrared fluorescence from this compound only weakly compared to normal tissue, while normal tissue emitted near-infrared fluorescence. Based on these findings, the inventors completed the present invention.

[0017] Specifically, the inventors administered a cyclodextrin-bound indocyanine compound that emits near-infrared fluorescence by near-infrared light irradiation intravenously (systemically) or intravesically to bladder cancer model mice for the purpose of distinguishing cancer, such as bladder cancer tissue, from normal tissue, and irradiated near-infrared light. As a result, it was found that normal tissue in the bladder does not emit near-infrared fluorescence derived from this compound, while cancer tissue emits near-infrared fluorescence. In addition, the inventors intravenously administered (systemically) a cyclodextrin-bound indocyanine compound to model mice having gastric cancer, peritoneal cancer derived from renal cell carcinoma or peritoneal seeding derived from gastric cancer, and esophageal cancer, and irradiated near-infrared light, and found that these cancer tissues emit stronger near-infrared fluorescence than the surrounding normal tissue. Further, the inventors intravenously administered (systemically) a cyclodextrin-bound indocyanine compound to model mice having renal cancer, lung cancer derived from renal cell carcinoma, and liver cancer derived from renal cell carcinoma, and irradiated near-infrared light, and found that the emission of near-infrared fluorescence from these cancer tissues is weaker than the emission of near-infrared fluorescence from the surrounding normal tissue, and that cancer tissue and normal tissue can be distinguished. In addition, the inventors administered a cyclodextrin-bound indocyanine compound to a living body and acquired a near-infrared fluorescence image of the kidney, thereby extracting abnormal sites of renal cells at the micro level and abnormal sites of renal tissue at the macro level. Furthermore, the inventors locally administered a cyclodextrin-bound indocyanine compound to a desired cancer tissue, such as colorectal cancer, breast cancer 、 skin cancer, head and neck cancer, and brain tumor, irradiated near-infrared light, and found that the cancer tissue emits near-infrared fluorescence when the compound accumulates in the cancer tissue.

[0018] Although not limited thereto, the present invention includes at least the following aspects. [1] The following formula:

[0019] [Chemical formula] [In the formula, the two Qs can be selected independently and may be the same or different, Q is *l-(CH2)a-CO-NH-(CH2)b-*3, where a is an integer between 2 and 6, b is an integer between 2 and 6, where *1 is the N of the indocyanine skeleton, and *3 is the part that replaces one of the three OH groups of any D-glucose constituting the cyclodextrin with -O- and is bonded.] R1 to R23 are each independently a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxyl group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphoric acid group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle, and each of R1 to R23 may be unsubstituted or substituted with a substituent, and if the substituent is a carboxylic acid, a sulfonic acid, or a phosphoric acid, the hydrogen ion may dissociate at the substituent and be replaced by a metal ion. R8 and R9 may together form a cyclic structure of CH2, CH2CH2, CH2CH2CH2 or CH2CH2CH2CH2, and one or more hydrogen atoms of the cyclic structure may be substituted with an aryl group, halogen atom, alkoxy group, amino group, carboxyl group, formyl group, sulfonyl group, sulfonic acid group, phosphate group, alkyloxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, or heterocycle. A cancer contrast-enhancing composition containing a cyclodextrin-bound indocyanine compound represented by or a pharmaceutically acceptable salt thereof. [2] The cyclodextrin-bound indocyanine compound is given by the following formula:

[0020] [ka] [In the formula, m, n, p, and q are each independently integers between 2 and 6, r is an integer between 5 and 7, s is an integer between 0 and 4, and R is a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxy group, amino group, carboxyl group, formyl group, sulfonyl group, sulfonic acid group, alkyloxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, or heterocycle] The composition according to [1], which is a compound represented by or a pharmaceutically acceptable salt thereof. [3] The cyclodextrin-bound indocyanine compound is given by the following formula:

[0021] [ka] [In the formula, m and n are each independently integers between 2 and 6, s is an integer between 0 and 4, and R is a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxy group, amino group, carboxyl group, formyl group, sulfonyl group, sulfonic acid group, alkyloxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, or heterocycle.] The composition according to [1] or [2], wherein the compound shown is or a pharmaceutically acceptable salt thereof. [4] The cyclodextrin-bound indocyanine compound is given by the following formula:

[0022] [ka] A composition according to any one of [1] to [3], comprising a compound represented by or a pharmaceutically acceptable salt thereof. [5] A liquid composition as described in any of [1] to [4]. [6] A composition according to any one of [1] to [5], for contrast imaging of bladder cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, peritoneal cancer, peritoneal dissemination, or esophageal cancer. [7] A composition according to any one of [1] to [5], for contrast imaging of breast cancer, colorectal cancer, skin cancer, head and neck cancer, or brain tumor. [8] A composition according to any one of [1] to [6] for systemic administration. [9] A composition according to any one of [1] to [5] and [7], for topical administration.

[10] A composition according to any one of [1] to [5], for contrast imaging of bladder cancer and for intravesical administration via the urethra.

[11] A composition according to any one of [1] to [5], for contrast imaging of bladder cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, peritoneal cancer, peritoneal dissemination, or esophageal cancer, and used by systemic administration.

[12] A composition according to any one of [1] to [5], for contrast imaging of breast cancer, colorectal cancer, skin cancer, head and neck cancer, or brain tumor, and used by local administration.

[13] A method of cancer imaging comprising administering a composition described in any of [1] to [5].

[14] A cyclodextrin-conjugated indocyanine compound or a pharmaceutically acceptable salt thereof, as described in any of [1] to [4], for contrast imaging of cancer.

[15] Use of any of the cyclodextrin-conjugated indocyanine compounds or pharmaceutically acceptable salts thereof described in [1] to [4] for contrast imaging of cancer.

[16] Use of a cyclodextrin-conjugated indocyanine compound or a pharmaceutically acceptable salt thereof as described in any of [1] to [4] for the preparation of a cancer contrast composition.

[0023] Furthermore, "subject" refers to a human or other animal requiring cancer contrast imaging, and in one aspect, it refers to a human requiring cancer contrast imaging.

[0024] The cyclodextrin-bound indocyanine compounds according to the present invention can encapsulate at least a portion of the naphthyl moiety of indocyanine by the cyclodextrin within the molecule. These compounds may reach an isomerized equilibrium state in aqueous solution, potentially resulting in an equilibrium state between the encapsulated and non-encapsulated forms. [Effects of the Invention]

[0025] According to the present invention, it becomes possible to specifically identify or distinguish cancerous tissue by irradiation with near-infrared light. Specifically, according to the present invention, it becomes possible to distinguish tumor tissues, including bladder cancer, kidney cancer, stomach cancer, liver cancer, lung cancer, peritoneal dissemination, peritoneal cancer, esophageal cancer, colorectal cancer, breast cancer, skin cancer, head and neck cancer, and brain tumors, based on the observation of near-infrared fluorescence emitted by near-infrared light irradiation.

[0026] When the compound according to the present invention is administered to a target, the compound remains in the cancer tissue. Therefore, by administering the compound according to the present invention systemically to a target with cancer such as bladder cancer, gastric cancer, peritoneal dissemination, peritoneal cancer, or esophageal cancer, or by administering it locally to cancer tissue such as colorectal cancer, breast cancer, skin cancer, head and neck cancer, or brain tumor, the cancer tissue will emit near-infrared fluorescence upon irradiation with near-infrared light.

[0027] In other words, the cancer contrast-enhancing composition according to the present invention, when administered into the tumor of an individual with cancer, exhibits higher tumor retention selectivity and lower background levels in normal tissue compared to indocyanine green, a known near-infrared fluorescent contrast agent, and can be used as a fluorescence contrast method that enables accurate tumor location identification during cancer surgery.

[0028] Furthermore, by administering the compound according to the present invention to a subject with certain types of cancer, such as kidney cancer, liver cancer, or lung cancer, it becomes possible to distinguish between cancerous tissue and normal tissue by near-infrared light irradiation. In other words, when the compound according to the present invention is administered systemically to a subject, the near-infrared fluorescence emitted by certain types of cancerous tissue becomes weaker than the near-infrared light emitted from the surrounding normal tissue, making it possible to distinguish between cancerous tissue and normal tissue, and enabling accurate identification of the tumor location during cancer surgery, etc.

[0029] In one embodiment, the present invention can provide identification guide technology for surgical resection of cancerous tissue and the like. [Brief explanation of the drawing]

[0030] [Figure 1]Figure 1 shows a photograph of the bladder one day after administering a cancer contrast-enhancing composition containing compound III via tail vein to MB49 mice with bladder cancer (left: under white light, right: near-infrared fluorescence image). [Figure 2] Figure 2 shows a photograph of the bladder of a normal mouse one day after administering a cancer contrast-enhancing composition containing compound III via tail vein (left: under white light, right: near-infrared fluorescence image). [Figure 3] Figure 3 shows images of the bladder of MB49 mice with bladder cancer after administration of a cancer contrast-enhancing composition containing compound III, followed by bladder irrigation with physiological saline 30 minutes later (left: under white light, right: near-infrared fluorescence image). [Figure 4] Figure 4 shows images of the bladder of a normal mouse after administration of a cancer contrast-enhancing composition containing compound III, followed by bladder irrigation with physiological saline 30 minutes later (left: under white light, right: near-infrared fluorescence image). [Figure 5] Figure 5 shows images of cancerous tissue extracted by near-infrared fluorescence observation one day after administering a cancer contrast-enhancing composition containing compound III to MB49 mice with bladder cancer via tail vein (left: under white light, right: near-infrared fluorescence image). [Figure 6] Figure 6 shows images of the bladder one day after administering a cancer contrast-enhancing composition containing compound IV via tail vein to MB49 mice with bladder cancer (left: under white light, right: near-infrared fluorescence image). [Figure 7] Figure 7 shows a photograph of the bladder of a normal mouse one day after administering a cancer contrast-enhancing composition containing compound IV via tail vein (left: under white light, right: near-infrared fluorescence image). [Figure 8] Figure 8 shows images of the bladder of MB49 mice with bladder cancer after administration of a cancer contrast-enhancing composition containing compound IV and subsequent bladder irrigation with physiological saline 10 minutes later (left: under white light, right: near-infrared fluorescence image). [Figure 9]Figure 9 shows images of the bladder of a normal mouse after administration of a cancer contrast-enhancing composition containing compound IV, followed by bladder irrigation with physiological saline 10 minutes later (left: under white light, right: near-infrared fluorescence image). [Figure 10] Figure 10 shows images of cancerous tissue extracted by near-infrared fluorescence observation one day after administering a cancer contrast-enhancing composition containing compound IV to MB49 mice with bladder cancer via tail vein (left: under white light, right: near-infrared fluorescence image). [Figure 11] Figure 11 shows macroscopic near-infrared fluorescence images of the surface of the kidneys of normal rats (top row) and the surface of pre- and malignant kidneys (bottom row) of iron-nitrilotriacetate-induced tumors at 30 minutes, 3 hours, and 24 hours after administration of a cancer contrast-enhancing composition containing compound III to normal rats and rats administered iron-nitrilotriacetate intraperitoneally. The magnified image at the bottom is a near-infrared fluorescence image of an iron-nitrilotriacetate-induced tumor kidney 3 hours after administration of the cancer contrast-enhancing composition containing compound III. The fluorescence intensity has been adjusted to improve visibility of the fluorescence images. [Figure 12] Figure 12 shows macroscopic near-infrared fluorescence images of cross-sections of normal rat kidneys (top row) and cross-sections of pre- and malignant kidneys (bottom row) of iron-nitrilotriacetate-induced tumors, at 30 minutes, 3 hours, and 24 hours after administration of a cancer contrast-enhancing composition containing compound III to normal rats and rats administered iron-nitrilotriacetate intraperitoneally. The magnified image at the bottom is a near-infrared fluorescence image of an iron-nitrilotriacetate-induced tumor kidney 3 hours after administration of the cancer contrast-enhancing composition containing compound III. Fluorescence intensity has been added to improve visibility of the fluorescence images. [Figure 13] Figure 13 shows micro-near-infrared fluorescence images of kidney tissue sections (renal cortex region) taken 3 hours after administration of a cancer contrast-enhancing composition containing compound III to pre-tumor / tumor-developing rats induced by iron-nitrilotriacetate (top: bright-field, middle: near-infrared fluorescence, bottom: merged). Fluorescence intensity has been arbitrarily adjusted for better visibility of the fluorescence images. The symbols in the figure are as follows: A: Casts formed within the tubular lumen (early stage of renal cyst) B, C, D: Distal tubule [Figure 14]Figure 14 shows near-infrared fluorescence images obtained by administering a cancer contrast-enhancing composition containing compound III or indocyanine green (ICG) into tumors of a 4T1 mouse syngeneic orthotopic breast cancer model (top: 30 minutes, middle: 60 minutes, bottom: 3 hours). [Figure 15] Figure 15 shows near-infrared fluorescence images of tumors in a CT26 mouse syngeneic colorectal cancer subcutaneous transplant model after administration of a cancer contrast-enhancing composition containing compound III (top: 30 minutes, middle: 60 minutes, bottom: 120 minutes). [Figure 16] Figure 16 shows near-infrared fluorescence images obtained by administering a cancer contrast-enhancing composition containing compound III into a tumor of a B16F1 mouse syngeneic orthotopic melanoma model (top: 30 minutes later, bottom: 60 minutes later). [Figure 17] Figure 17 shows a near-infrared fluorescence image of a tumor isolated from a mouse and picked up with tweezers, obtained by fluorescence imaging performed during the 4T1 tumor resection procedure. [Figure 18] Figure 18 shows a color image (left) and a near-infrared fluorescence image (right) of a cross-section of the lung 10 minutes after administration of a cancer contrast-enhancing composition containing compound III via tail vein to mice with lung cancer. In the near-infrared fluorescence image, white areas indicate strong fluorescence, and the areas enclosed by the frame are cancerous sites (scale bar: 10 mm). [Figure 19] Figure 19 shows a color image (left) and a near-infrared fluorescence image (right) of the liver after administering a cancer contrast-enhancing composition containing compound III via tail vein to mice with liver cancer. White areas in the near-infrared fluorescence image indicate fluorescence, and the areas enclosed by frames in both the color and near-infrared fluorescence images are liver cancer sites (scale bar: 10 mm). [Figure 20] Figure 20 shows near-infrared fluorescence images of the time course after administering a cancer contrast-enhancing composition containing compound III via tail vein to mice subcutaneously transplanted with human gastric cancer cells. White areas in the near-infrared fluorescence images indicate fluorescence. The circles in the images indicate cancer tissue (kidney). [Figure 21]Figure 21 shows a color image (left) and a near-infrared fluorescence image (right) of the intraperitoneal cavity after administering a cancer contrast-enhancing composition containing compound III via the tail vein to a peritoneal cancer model mouse. White areas in the near-infrared fluorescence image indicate fluorescence. The areas enclosed by frames in the color image and near-infrared fluorescence image are peritoneal cancers (kidney: kidney, bladder: bladder, scale bar: 10 mm). [Figure 22] Figure 22 shows color images (left) and near-infrared fluorescence images (right) of the intraperitoneal cavity 10 minutes after administration of a cancer contrast-enhancing composition containing compound III via tail vein to normal mice. White areas in the near-infrared fluorescence image indicate fluorescence (scale bar: 10 mm). [Figure 23] Figure 23 shows a color image (left) and a near-infrared fluorescence image (right) of the intraperitoneal cavity 10 minutes after administration via the tail vein to a peritoneal dissemination model mouse in which human gastric cancer cells were transplanted intraperitoneally with a cancer contrast-enhancing composition containing compound III. In the near-infrared fluorescence image, white indicates fluorescence, and the area within the dotted line in the image indicates solid tumor tissue that could be visually confirmed (scale bar: 10 mm). [Figure 24] Figure 24 shows a color image (left) and a near-infrared fluorescence image (right) of the intraperitoneal cavity 10 minutes after administration of a cancer contrast-enhancing composition containing compound III via tail vein to mice with renal cancer. White areas in the near-infrared fluorescence image indicate fluorescence, and the areas enclosed by frames in the image represent regions of cancerous tissue that could be visually identified (kidney: kidney, scale bar: 10 mm). [Figure 25] Figure 25 shows a color image (left) and a near-infrared fluorescence image (right) 30 seconds after administration of a cancer contrast-enhancing composition containing compound III to a human esophageal cancer subcutaneous transplantation model mouse via tail vein. The area enclosed by the circular frame indicates cancer tissue (scale bar: 10 mm). The fluorescence within the oval frame indicates near-infrared fluorescence from the left and right kidneys, and the white areas in the near-infrared fluorescence image indicate fluorescence. [Figure 26] Figure 26 shows near-infrared fluorescence images of a cancer contrast-enhancing composition containing compound III administered into a tumor in a human tongue cancer subcutaneous transplant model (top: immediately after administration, bottom: 50 minutes later). [Figure 27] Figure 27 shows near-infrared fluorescence images (1 hour after administration) of a cancer contrast-enhancing composition containing compound III administered into a tumor in a human neuroblastoma subcutaneous transplant model. [Figure 28] Figure 28 shows near-infrared fluorescence images (1 hour after administration) of a cancer contrast-enhancing composition containing compound III administered into a tumor in a human glioblastoma subcutaneous transplant model. [Modes for carrying out the invention]

[0031] The present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0032] In one embodiment, the present invention is a composition for imaging cancer, and the present invention is a cancer contrast-enhancing pharmaceutical composition used in the pharmaceutical field. The cancer contrast-enhancing composition according to the present invention is given by the following formula:

[0033] [ka] It contains a cyclodextrin-bound indocyanine compound represented by or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as compound A). In formula A, the two Qs can be selected independently and may be the same or different, Q is *l-(CH2)a-CO-NH-(CH2)b-*3, a is an integer between 2 and 6, b is an integer between 2 and 6, *1 is the N of the indocyanine skeleton, and *3 is the part that replaces one of the three OH groups of any D-glucose constituting the cyclodextrin with -O- and is bonded. R1 to R23 are each independently a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxyl group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, a phosphoric acid group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle, and each of R1 to R23 may be unsubstituted or substituted with a substituent, and if the substituent is a carboxylic acid, a sulfonic acid, or a phosphoric acid, the hydrogen ion may dissociate at the substituent and be replaced by a metal ion. R8 and R9 may together form a cyclic structure of CH2, CH2CH2, CH2CH2CH2, or CH2CH2CH2CH2, where one or more hydrogen atoms of the cyclic structure may be substituted with an aryl group, halogen atom, alkoxy group, amino group, carboxyl group, formyl group, sulfonyl group, sulfonic acid group, phosphoric acid group, alkyloxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, or heterocycle. In one embodiment, the number of carbon atoms in R1 to R23 in formula A is independently 1 to 5, or 1 to 3. Here, when R1 to R23 are substituted with a carboxylic acid, sulfonic acid, or phosphoric acid, the hydrogen ions of the carboxylic acid, sulfonic acid, or phosphoric acid may be substituted with sodium, potassium, or magnesium.

[0034] "Alkyl group" refers to a linear or branched alkyl group having 1 to 20 carbon atoms, which may have substituents. Examples include linear groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosanil, or groups that are linked in a branched manner.

[0035] Examples of "aryl groups" include aromatic hydrocarbons with 6 to 20 carbon atoms, such as phenyl and naphthyl.

[0036] In the present invention, "alkoxyl group" can refer to, for example, alkoxyl groups having 1 to 20 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methixiethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, and methoxyethoxyethoxy groups, which are bonded in a linear or branched manner.

[0037] An "alkyloxycarbonyl group" is a group represented by the alkyl group -OC(=O)-. An "aryloxycarbonyl group" is a group represented by the aryl group -OC(=O)-. An "alkylcarbonyl group" is a group represented by the alkyl group -C(=O)-. An "arylcarbonyl group" is a group represented by the aryl group -C(=O)-.

[0038] "Heterocycle" refers to a 5-7 member unsaturated heterocycle or saturated heterocycle containing 1-4 heteroatoms selected from nitrogen, oxygen, or sulfur atoms, or a bicyclic heterocycle fused with a benzene ring or other heterocycle. Examples of aromatic heterocycles include pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, triazole, thiophene, thiopyran, furan, pyran, dioxolane, thiazole, isothiazole, thiadiazole, thiazine, oxazole, isoxazole, oxadiazole, dioxazole, oxazine, and oxadi Examples of saturated heterocycles include azines and dioxazines, while saturated heterocycles include pyrrolidine, piperidine, piperazine, morpholine, and thiomorpholine. Condensed aromatic heterocycles include indole, isoindole, indazole, quinoline, quinazoline, quinoxaline, isoquinoline, benzimidazole, benzothiophene, benzothiazole, benzofuran, benzofurazan, imidazopyridine, imidazopyrazine, pyridopyridine, phthalazine, naphthyridine, indidine, purine, quinolidine, cinnoline, isocoumarin, and chroman.

[0039] "Halogen atom" means a fluoro, chloro, bromo, or iodine atom. In one embodiment, it is a fluoro, chloro, or bromo atom, and in another embodiment, it is a chloro atom.

[0040] A "substituent" may consist of one to three identical or different substituents. "Substituents on the amino group in the case of secondary, tertiary, and quaternary nucleotides" include halogen atoms, alkyl groups, etc.

[0041] The cyclodextrin-bound indocyanine compound, which is the active ingredient of the composition of the present invention, is a cyclodextrin-bound indocyanine compound formed by the covalent bonding of indocyanines and cyclic sugar chain cyclodextrin, and can be in a state in which at least a portion of the naphthyl group of indocyanine is encapsulated in the cavity of cyclodextrin. Furthermore, the indocyanine group may have substituents as long as the naphthyl group of indocyanine is encapsulated in the cavity of cyclodextrin and emits near-infrared fluorescence.

[0042] For at least a portion of the naphthyl group of indocyanine to be encapsulated in the cavity of cyclodextrin, a spacer Q may be used, and the indocyanines and the cyclic sugar chain cyclodextrin may be covalently bonded via the spacer. Here, by adjusting the length of the spacer, it is possible to control the degree of encapsulation of the naphthyl group of indocyanine into the cavity of cyclodextrin. Considering the ease of encapsulation by cyclodextrin, the spacer Q may have a structure between the nitrogen atom in the structure corresponding to indocyanine and the oxygen atom in the structure corresponding to cyclodextrin, with a number of atoms between 7 and 9.

[0043] The cyclodextrin in formula A is not particularly limited and includes, for example, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, and the cyclodextrin may have substituents. In one embodiment, the cyclodextrin in formula A is β-cyclodextrin. The cyclodextrin may also have substituents.

[0044] The compound used in the cancer contrast-enhancing composition according to the present invention can encapsulate at least a portion of the naphthyl moiety of indocyanine by intramolecular cyclodextrin. The compound according to the present invention can reach an isomerization equilibrium state in aqueous solution, and can exist in equilibrium between an inclusion-type and an uninclusion-type.

[0045] The compound according to the present invention exhibits fluorescence in the near-infrared region (700 nm to 2500 nm), for example, around 800 to 830 nm. By administering it to a living organism and acquiring near-infrared fluorescence images, it is possible to extract abnormal areas in biological tissue at both the micro and macro levels.

[0046] In one embodiment, the cyclodextrin-bound indocyanine compound according to the present invention is given by the following formula:

[0047] [ka] The compound represented by or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as compound B). In formula B, m, n, p, and q are each independently integers between 2 and 6, r is an integer between 5 and 7, s is an integer between 0 and 4, and R is a hydrogen atom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, an amino group, a carboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, or a heterocycle. In one embodiment, the number of carbon atoms in R in formula B is 1 to 5, and may be 1 to 3.

[0048] In one embodiment, the cyclodextrin-bound indocyanine compound according to the present invention is given by the following formula:

[0049] [ka] The compound represented by or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as compound C). In formula C, m and n are each independently integers between 2 and 6, s is an integer between 0 and 4, and R is a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxy group, amino group, carboxyl group, formyl group, sulfonyl group, sulfonic acid group, alkyloxycarbonyl group, aryloxycarbonyl group, alkylcarbonyl group, arylcarbonyl group, or heterocycle. In one embodiment, the number of carbon atoms in R in formula C is 1 to 5, and may be 1 to 3.

[0050] In one embodiment, the compound according to the present invention or a pharmaceutically acceptable salt thereof is such that R in formula B or formula C is an alkoxy group.

[0051] In one embodiment of the present invention, the cancer contrast-enhancing composition according to the present invention contains a compound represented by formula I or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as compound I).

[0052] [ka] Furthermore, in one embodiment of the present invention, the cancer contrast-enhancing composition according to the present invention contains a compound represented by formula II or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as compound II).

[0053] [ka]

[0054] In one aspect, the cancer contrast-enhancing composition according to the present invention is for systemic administration.

[0055] In one aspect, the cancer contrast-enhancing composition according to the present invention is a bladder cancer contrast-enhancing composition.

[0056] In one embodiment, the cancer contrast-enhancing composition according to the present invention is a contrast-enhancing composition for renal cancer.

[0057] In one aspect, the cancer contrast-enhancing composition according to the present invention is a lung cancer contrast-enhancing composition.

[0058] In one embodiment, the cancer contrast-enhancing composition according to the present invention is a liver cancer contrast-enhancing composition.

[0059] In one aspect, the cancer contrast-enhancing composition according to the present invention is a gastric cancer contrast-enhancing composition.

[0060] In one embodiment, the cancer contrast-enhancing composition according to the present invention is a peritoneal cancer contrast-enhancing composition.

[0061] In one embodiment, the cancer contrast-enhancing composition according to the present invention is a peritoneal dissemination contrast-enhancing composition.

[0062] In one aspect, the cancer contrast-enhancing composition according to the present invention is a contrast-enhancing composition for esophageal cancer.

[0063] In one aspect, the cancer contrast-enhancing composition according to the present invention is for intravesical administration via the urethra.

[0064] In one aspect, the cancer contrast-enhancing composition according to the present invention is for local administration.

[0065] In one aspect, the cancer contrast-enhancing composition according to the present invention is a breast cancer contrast-enhancing composition.

[0066] In one aspect, the cancer contrast-enhancing composition according to the present invention is a colorectal cancer contrast-enhancing composition.

[0067] In one aspect, the cancer contrast-enhancing composition according to the present invention is a skin cancer contrast-enhancing composition.

[0068] In one embodiment, the cancer contrast-enhancing composition according to the present invention is a composition for head and neck cancer contrast-enhancing.

[0069] In one aspect, the cancer contrast-enhancing composition according to the present invention is a brain tumor cancer contrast-enhancing composition.

[0070] In one embodiment of the present invention, the cancer contrast-enhancing composition according to the present invention contains the chloride of compound I (compound III).

[0071] In one embodiment of the present invention, the cancer contrast-enhancing composition according to the present invention contains the chloride of compound II (compound IV).

[0072] In one embodiment of the present invention, compound A, B, C, I, II, III, or IV is used for the production of a cancer contrast-enhancing composition according to the present invention.

[0073] In one embodiment of the present invention, the use of compound A, B, C, I, II, III, or IV for cancer contrast imaging is provided.

[0074] In one aspect of the present invention, the cancer contrast-enhancing composition according to the present invention is an injectable solution.

[0075] Compounds according to the present invention or pharmaceutically acceptable salts thereof can be obtained, for example, based on the method described in U.S. Patent Publication 2012 / 0302881.

[0076] "Pharmaceutically acceptable salts" refer to acid addition salts of compounds, which can be obtained by conventional salt-making reactions. Specifically, these include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyl tartaric acid, ditoluyl tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid.

[0077] The cancer contrast-enhancing composition according to the present invention contains the compound according to the present invention or a pharmaceutically acceptable salt thereof, but may also contain pharmaceutically acceptable excipients, etc. The cancer contrast-enhancing composition according to the present invention can be prepared by commonly used methods using pharmaceutically acceptable excipients, carriers, etc.

[0078] The cancer contrast-enhancing composition according to the present invention is not particularly limited in its route of administration and may be administered systemically or locally, parenterally or orally. The route of administration may be appropriately selected depending on the target of administration and cancer tissue, for example, bladder cancer can be visualized by intravesical administration via the urethra. The cancer contrast-enhancing composition according to the present invention can be administered to the target by, for example, systemic intravenous administration, intra-articular, intramuscular, or intra-cancer tissue local administration. For example, the dosage form of the cancer contrast-enhancing composition according to the present invention is not particularly limited, but one embodiment is that it is a liquid formulation. Depending on the dosage form, the cancer contrast-enhancing composition according to the present invention may optionally contain pharmaceutically acceptable additives such as suspensions, solubilizers, isotonic agents, preservatives, anti-adsorption agents, analgesics, sulfur-containing reducing agents, and antioxidants, in addition to pharmaceutically acceptable excipients and carriers.

[0079] Injectable preparations for parenteral administration may contain, for example, sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Aqueous solvents include, for example, distilled water for injection or physiological saline. Non-aqueous solvents include, for example, alcohols such as ethanol. Such compositions may further contain isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, or solubilizers. These are sterilized, for example, by filtration through a bacterial retention filter, formulation with a bactericide, or irradiation. Alternatively, sterile solid compositions may be prepared and dissolved or suspended in sterile water or a sterile solvent for injection before use.

[0080] The dosage should be determined appropriately on an individual basis, taking into account the symptoms, age, sex, and size of the tumor of the patient. When administered intravenously, for example, the daily dose is approximately 0.0001 to 10 mg / kg per body weight in a preferred configuration, administered once or multiple times a day. When administered locally to cancer, for example, approximately 0.001 to 100 mg / kg per body weight can be administered once or multiple times.

[0081] Although it varies depending on the route of administration, dosage form, site of administration, and type of excipients and additives, the contrast-enhancing composition according to the present invention may contain 0.01 to 99% by weight of the compound according to the present invention as an active ingredient, and in one embodiment, it may contain 0.01 to 50% by weight of the compound according to the present invention.

[0082] Depending on the type of cancer, the cancer contrast-enhancing pharmaceutical composition according to the present invention may be advantageous for systemic administration or for local administration.

[0083] One possible timing of administration is preoperative administration or intraoperative administration. Another possible timing of administration is intraoperative administration.

[0084] In one aspect, the present invention relates to a method for contrast-enhancing cancer. In this invention, an effective amount of a cancer contrast-enhancing composition is administered to a target, and near-infrared light is irradiated onto the desired cancer using a near-infrared fluorescence imaging device. By detecting the near-infrared fluorescence emitted from the compound according to the present invention, it is possible to enhance the desired cancer.

[0085] The cancers that can be visualized by the present invention are not particularly limited, but for example, cancers of the kidneys, bladder, ureters, urethra, esophagus, stomach, small intestine, large intestine, mammary gland, skin, liver, lung, peritoneum, head and neck, and brain can be visualized. In other words, the present invention makes it possible to image kidney cancer, bladder cancer, esophageal cancer, stomach cancer, large intestine cancer, breast cancer, skin cancer, lung cancer, liver cancer, peritoneal cancer, peritoneal dissemination, head and neck cancer, brain tumors, etc. with near-infrared light. These cancers also include cancers that have metastasized from cancers originating from other organs and developed in the organ in question. For example, lung cancer includes lung cancer that originated from kidney cancer. Here, brain tumor refers to a malignant tumor (cancer) that occurs in the brain, and examples include neuroblastoma and glioblastoma. Head and neck cancer refers to a malignant tumor (cancer) that occurs in the head and neck, and an example is tongue cancer.

[0086] Systemic administration allows for contrast imaging of, for example, bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, peritoneal cancer, peritoneal dissemination, or esophageal cancer.

[0087] Local administration allows for contrast enhancement of, for example, breast cancer, colorectal cancer, skin cancer, head and neck cancer, or brain tumors.

[0088] In one aspect, cancer is a primary cancer that first develops in the organ in question.

[0089] In one form, cancer is a metastatic cancer that develops in a given organ after a cancer that originated in another organ has spread there.

[0090] Typical contrast-enhancing methods used at the experimental level are as follows: 1. Creation of tumor-bearing mice For example, we can create tumor-bearing mice using a standard method, referring to "Cancer - Creation and Utilization of Disease Models" (Takuro Nakamura, L.I.C., 2012). 2. In the case of bladder cancer A saline solution of the compound according to the present invention is administered intravenously to mice, and approximately one day later, near-infrared light is irradiated into the bladder using a near-infrared fluorescence imaging device to detect the near-infrared fluorescence emitted from the compound. The dosage of the compound is, for example, 0.01 to 10 mg per 1 kg of mouse, or in another embodiment, 0.1 to 1 mg. 3. In the case of bladder cancer A saline solution containing the compound according to the present invention is administered to the bladder of a mouse via the urethra. After approximately 10 minutes to 2 hours, the bladder is washed with water or saline solution, and near-infrared light is irradiated onto the bladder using a near-infrared fluorescence imaging device to detect near-infrared fluorescence. The saline solution containing the compound according to the present invention is used in a volume that can be filled into the target bladder. For example, the saline solution containing the compound according to the present invention contains the compound according to the present invention at a concentration of 0.00001 to 1 mg / mL, and in another embodiment, at a concentration of 0.001 to 0.1 mg / mL. The number of bladder washes can be varied according to the concentration of the injected compound. Bladder washes involve filling and draining the entire bladder with water or saline solution, exchanging the bladder fluid 5 to 10 times. 4. In the case of other cancers A saline solution of the compound according to the present invention, 0.05 to 0.5 mL, is administered locally to the tumors of tumor-bearing mice, and then the desired tumors are irradiated with near-infrared light using a near-infrared fluorescence imaging device to detect the near-infrared fluorescence emitted from the compound. The saline solution containing the compound according to the present invention contains, for example, the compound according to the present invention at a concentration of 0.00001 to 1 mg / mL.

[0091] After intravenously administering a saline solution of the compound according to the present invention to a mouse, near-infrared fluorescence is detected 30 seconds to 120 minutes later by irradiating the body surface from outside the skin, into the abdominal cavity after laparotomy, into the chest after thoracotomy, or in the organ to be observed, using a near-infrared fluorescence imaging device. The dosage of the compound is, for example, 0.00001 to 10 mg per kg of body weight, or in another embodiment, 0.01 to 1 mg.

[0092] Furthermore, the following are other embodiments of the present invention.

[0093] In one embodiment, the present invention is a technology for identifying cancerous tissue, and the present invention encompasses a method and apparatus for identifying cancerous tissue. Because the present invention makes it possible to accurately identify cancerous tissue, the present invention can be applied as a guide technology in surgery and the like. In one embodiment of the present invention, according to the present invention, objective resection surgery that does not depend on the surgeon's judgment ability to identify the cancerous site becomes possible.

[0094] In one embodiment, the present invention is a surgical support device applicable to various surgeries. The device according to the present invention may be equipped with an imaging device, and is expected to enable automated robotic resection surgery, for example, by determining the resection site using artificial intelligence (AI) utilizing a computer.

[0095] In one aspect, the present invention is the use of a compound according to the present invention for producing a cancer contrast-enhancing composition. In another aspect, the present invention is a compound according to the present invention for producing a cancer contrast-enhancing composition. In yet another aspect, the present invention is the use of a compound according to the present invention for cancer contrast.

[0096] The apparatus according to the present invention may include means for irradiating a target to which the compound according to the present invention has been administered with excitation light, and means for measuring fluorescence intensity.

[0097] The excitation light irradiation means is a means of irradiating the administered compound of the present invention with excitation light of a wavelength capable of producing fluorescence. The wavelength of the excitation light to be irradiated can be limited to an appropriate range. By limiting the wavelength to the narrowest possible range, separation of fluorescence and excitation light can be reliably achieved. Wavelength limitation can be achieved by selecting a light source that emits light of an appropriate wavelength or by limiting the wavelength with a filter.

[0098] The manner of excitation light irradiation is not particularly limited as long as the generated fluorescence can be measured by the fluorescence intensity measuring means described later. For example, the excitation light can be continuous, pulsed, or of varying intensity. When varying the intensity, the intensity of the excitation light can be modulated by irradiating pulses of excitation light at predetermined intervals. It is desirable to modulate the intensity of the excitation light by employing pulse amplitude modulation.

[0099] The excitation light is delivered to the area to be irradiated using an appropriate optical system. The area to be irradiated is the area in the body where cancerous tissue is suspected to be present. The range of irradiation with excitation light is not particularly limited and is determined as needed. For example, irradiating a narrow area allows for precise measurement in that narrow area.

[0100] Furthermore, it is preferable to perform the irradiation of excitation light by the excitation light irradiation means in a state that suppresses the influence of ambient light. For example, it is preferable to irradiate with excitation light in a dark place, or to irradiate with excitation light while the part to be irradiated with excitation light is covered from ambient light.

[0101] The fluorescence intensity measuring means is a means for measuring the intensity of fluorescence emitted from a site irradiated with excitation light by an excitation light irradiation means. It is preferable to measure the fluorescence intensity by excluding light other than fluorescence (ambient light, excitation light, etc.) through a filter that selectively transmits the emitted fluorescence.

[0102] When employing a method of irradiating excitation light with modulated intensity, the fluorescence intensity can be obtained by separating the component showing a change corresponding to the modulation from the measured light intensity. For example, when the excitation light intensity is modulated by pulse amplitude modulation, the fluorescence intensity can be separated by demodulating the light component that changes according to the intensity of the modulated pulse and measuring its intensity. Therefore, the influence of ambient light on the fluorescence intensity measurement results can be reduced.

[0103] The apparatus according to the present invention may include fluorescence imaging means and morphological imaging means, and may also include display means. According to the present invention, since the location where cancer tissue is present can be accurately identified, the apparatus according to the present invention can be suitably used as a guide device during surgery.

[0104] The fluorescence imaging means is a means of obtaining distribution data of the compound in a living organism by acquiring the intensity of fluorescence emitted by the compound according to the present invention, which has been excited by the excitation light irradiation means. In other words, this means is a means of acquiring distribution data that represents the distribution of the compound in the present invention in a part of a living organism as image data.

[0105] The fluorescence imaging means according to the present invention can be configured, for example, by combining a suitable optical system with an image sensor such as a CCD. The resolution of the acquired data is set to a value required depending on the purpose. When measuring fluorescence intensity, it is preferable to measure it by excluding light other than fluorescence (ambient light, excitation light, etc.) by passing it through a filter that can selectively transmit the emitted fluorescence.

[0106] The morphological imaging means is a method for obtaining morphological data of a part of a living organism by acquiring the intensity of light at wavelengths other than the fluorescence wavelength emitted by the compound according to the present invention. In other words, this means is a method for acquiring morphological data that represents the form of a part of a living organism as image data.

[0107] Morphological imaging can be constructed using an appropriate optical system and an image sensor such as a CCD. The resolution of the acquired data is set to the required value depending on the purpose. In this case, fluorescence emitted by the excitation light is not detected (or its detection sensitivity is reduced). The morphological imaging system can share most of the optical system with the aforementioned fluorescence imaging system, and a configuration can be adopted in which, in the optical path before finally introducing the light to the image sensor, light of the wavelength corresponding to fluorescence is guided to the fluorescence imaging system by a spectral prism or the like, and light of other wavelengths is guided to the morphological imaging system. The spectral prism can appropriately control the wavelength of light that is separated by appropriately forming a dichroic film or the like.

[0108] In this invention, the fluorescence imaging means and the morphological imaging means can be shared in a single imaging device. That is, the separation of fluorescence from other light may be mathematically separated after the image data has been created. Furthermore, distribution state data can be obtained as multiple image data from the surface of a part of the living organism toward the depth direction. Multiple image data in the depth direction can be obtained by moving the focal point in the optical system used in the fluorescence imaging means toward the depth direction. In addition, by employing an optical system with a changeable focal length as the excitation light irradiation means, the focal point can be moved to the interior of the living organism toward the depth direction instead of the surface of a part of the living organism, or the excitation light can be narrowed and irradiated onto a part of the living organism, so that fluorescence can be selectively generated not only on the surface of the living organism but also in the interior of the living organism toward the depth direction, and the circulation of that part can be visualized.

[0109] The display means is a means of displaying the distribution state of the compound of the present invention in a part of a living organism by superimposing distribution state data obtained by fluorescence imaging means onto morphological data obtained by morphological imaging means. If the fluorescence wavelength is not in the range of visible light, the fluorescence wavelength is converted to visible light of an appropriate wavelength and displayed. From the viewpoint of visualizing circulation, it is desirable to display the distribution state data with priority over the morphological data. In particular, areas where the distribution amount (i.e., fluorescence intensity) of the compound of the present invention is low (whether it is low or not is determined according to the purpose of visualizing circulation. For example, for the purpose of visualizing cancer tissue, it is areas that are not circulating, i.e., areas where fluorescence is not observed) are displayed in a way that makes them distinguishable from other areas. For example, these areas can be displayed in a different color from other areas or displayed by flashing. The superimposition of distribution state data and morphological data can be realized by computer logic, etc. The display of the data can be realized by a general display device. This display device can be placed between the living organism and the person performing the measurement, so that treatment can be performed on the living organism while viewing the display device.

[0110] Contrast enhancement includes normal enhancement and reverse enhancement. Normal enhancement means that the cyclodextrin-bound indocyanine compound according to the present invention remains in the cancer, and the near-infrared fluorescence emitted from the cancer tissue is stronger than that of the surrounding normal tissue, allowing the cancer tissue to be distinguished from surrounding normal cells. Reverse enhancement means that the cyclodextrin-bound indocyanine compound according to the present invention does not remain in the cancer compared to the surrounding normal tissue, and the near-infrared fluorescence emitted from the cancer tissue is weaker than that of the surrounding normal tissue, allowing the cancer tissue to be distinguished from surrounding normal tissue. Depending on the image processing method, reverse enhancement can be created as normal enhancement, and the opposite enhancement can also be created.

[0111] In one aspect, the present invention can be used to identify cancerous tissue. The cancers that can be identified in the present invention are not particularly limited, but examples include kidney cancer, bladder cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, skin cancer, lung cancer, liver cancer, peritoneal cancer, peritoneal dissemination, head and neck cancer, and brain tumors. The route of administration can be appropriately selected depending on the type of cancer. For example, when identifying bladder cancer, the compound according to the present invention can be administered systemically by intravenous administration, and the bladder can be irradiated with near-infrared light. By observing the near-infrared fluorescence emitted from the compound that has specifically accumulated in the bladder cancer tissue, the bladder cancer tissue can be identified. Furthermore, the compound according to the present invention can also be injected into the bladder via the urethra, and then, if necessary, the bladder can be washed and removed to remove compounds that are not adsorbed to the bladder cancer tissue. Moreover, in the present invention, bladder cancer tissue can also be identified by systemic and local administration of the compound according to the present invention.

[0112] In the present invention, the fluorescence intensity measuring means used for identifying cancerous tissue can be appropriately selected depending on the type of cancer, and which is suitable for measuring the site where the cancer has occurred. For example, in the case of bladder cancer, it is possible to identify bladder cancer tissue from the inside of the bladder using a near-infrared fluorescence cystoscope, or to identify bladder cancer tissue from the outside of the bladder using a near-infrared fluorescence observation device. [Examples]

[0113] A representative embodiment of the present invention will be specifically described by the following examples, but the present invention is not limited to the following examples and can be implemented with various modifications within the scope of the present invention. In this specification, unless otherwise specified, concentrations and the like are given on a weight basis, and numerical ranges are given including their endpoints.

[0114] In the example below, the chlorides of either Compound I or Compound II were used as the cyclodextrin-bound indocyanine compounds. These compounds exhibit fluorescence in the near-infrared region (700 nm to 2500 nm), particularly around 800 nm to 830 nm.

[0115] ■ Preparation of cyclodextrin-bound indocyanine compounds (1) Compound III (chloride of Compound I) 58 kg of methanol, 2.6 kg of dimethyl sulfoxide, 19 kg of water, 3-(2-carboxyethyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(2-carboxyethyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene]ethylidene}-2-methoxycyclohexa-1-en-1-yl]ethen-1-yl}-1,1-dimethyl-1H-benzo[e]indole-3-ium chloride, and 21-O-(3-aminopropyl)cyclomaltoheptaose (pure content: 21.5 kg) were mixed at approximately 20°C.

[0116] Next, 4.68 kg (3.98 kg pure) of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM) was added, washed with an aqueous methanol solution (6.8 kg methanol and 2.1 kg water), and stirred at approximately 20°C for approximately 2 hours. Then, 2.34 kg (1.99 kg pure) of DMT-MM and 0.726 kg of N-methylmorpholine were added and stirred at approximately 20°C for approximately 2 hours. Then, 2.34 kg (1.99 kg pure) of DMT-MM was charged at approximately 20°C. 84 kg of methanol was added dropwise over approximately 1 hour and stirred at approximately 20°C for approximately 16 hours. 250 kg of acetone was added dropwise over 2 hours, and the resulting suspension was stirred at internal temperature of approximately 20°C for approximately 19 hours. The filtrate was filtered, washed with an acetone-methanol mixture (58 kg acetone and 28 kg methanol), and then washed twice more with 84 kg of acetone. The filtrate was dried under reduced pressure at an ambient temperature of approximately 30°C to obtain 24.6 kg of green solid. This was dissolved in a 5 mM hydrochloric acid aqueous solution and purified by ODS column chromatography (ODS 130 kg, mobile phase: 5 mM hydrochloric acid aqueous solution → 30% methanol aqueous solution (v / v) → 40% methanol aqueous solution (v / v) → 80% methanol aqueous solution (v / v)). The target product was recovered, and methanol was removed under reduced pressure to obtain a concentrate.

[0117] Next, the entire concentrate was adsorbed onto an HP20SS column chromatograph (HP20SS 130 kg, mobile phase: 60% methanol aqueous solution (v / v) → 80% methanol aqueous solution (v / v)), and the active portion was collected by filtration. After concentrating the active portion under reduced pressure, it was filtered for clarification and then freeze-dried to obtain 8.62 kg of green solid. Of this, 4.9 kg was dissolved in a 1 mM hydrochloric acid aqueous solution and purified by ODS column chromatography (ODS 150 kg, mobile phase: 1 mM hydrochloric acid aqueous solution → 30% methanol aqueous solution (v / v) → 40% methanol aqueous solution (v / v)). The target product was recovered, and methanol was removed under reduced pressure to obtain a concentrate. Next, the entire concentrate was adsorbed onto an HP20SS column chromatograph (HP20SS 67.8 kg, mobile phase: 60% methanol aqueous solution (v / v) → 80% methanol aqueous solution (v / v)), and the active portion was collected and concentrated under reduced pressure to obtain a concentrate. Activated carbon was added and stirred, and the mixture was stirred at approximately 20°C for approximately 2 hours. The activated carbon was removed using a filter pre-coated with radiolight, and after further clarification filtration, freeze-drying was performed to obtain 1.88 kg of 3-(3-{[3-(cyclomaltomeptaose-21-O-yl)propyl]amino}-3-oxopropyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltomeptaose-21-O-yl)propyl]amino}-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene]ethylidene}-2-methoxycyclohexa-1-en-1-yl]ethen-1-yl}-1,1-dimethyl-1H-benzo[e]indole-3-ium chloride (compound III) as a green solid.

[0118] The physicochemical properties of the obtained compounds were as follows:

[0119] [Table 1]

[0120] (2) Compound IV (chloride of Compound II) Based on the method described in Mol. Pharm. 17, 2672-2681 (2020) (CD-NIR-1), the chloride of compound II (compound IV) was prepared.

[0121] ■Experiment 1 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound III (compound III concentration: 0.075 mg / mL) was administered into the tail vein of these mice. One day later, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened. Near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging system (Hamamatsu Photonics pde-neo, excitation wavelength: 760 nm, detection wavelength: fluorescence above 800 nm, the same applies hereafter) (Figure 1, right: near-infrared fluorescence image).

[0122] As a comparative example, a saline solution containing compound III was administered into the tail vein of normal mice (body weight approximately 20g). One day later, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened. Near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging device (pde-neo, Hamamatsu Photonics) (Figure 2).

[0123] Cancer tissue from bladder cancer model mice specifically exhibited near-infrared fluorescence (Figure 1, right: near-infrared fluorescence image), while normal tissue did not emit near-infrared fluorescence (Figure 2, right: near-infrared fluorescence image).

[0124] ■Experiment 2 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound III (compound III concentration: 0.075 mg / mL) was administered into the bladder of these mice via the urethra, and 30 minutes later, the bladder was irrigated five times with physiological saline via the urethra. Subsequently, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened, and near-infrared fluorescence observation was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo) (Figure 3, right: near-infrared fluorescence image).

[0125] As a comparative example, a saline solution containing compound III was administered to the bladder of a normal mouse (body weight approximately 20g) via the urethra, and 30 minutes later the bladder was washed five times with saline via the urethra. Subsequently, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened, and near-infrared fluorescence observation was performed using a near-infrared fluorescence imaging device (pde-neo, Hamamatsu Photonics, Ltd.) (Figure 4).

[0126] Cancer tissue from bladder cancer model mice specifically exhibited near-infrared fluorescence (Figure 3, right: near-infrared fluorescence image), while normal tissue did not emit near-infrared fluorescence (Figure 4, right: near-infrared fluorescence image).

[0127] ■Experiment 3 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound III (compound III concentration: 0.075 mg / mL) was administered into the tail vein of these mice. One day after administration, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened. Near-infrared fluorescence was observed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo), and the cancerous tissue areas that emitted near-infrared fluorescence were removed and observed for near-infrared fluorescence (Figure 5, right: near-infrared fluorescence image).

[0128] Since cancer tissue fluoresces when irradiated with near-infrared light, according to the present invention, tumor tissue could be appropriately removed by using the fluorescence emitted by the cancer tissue as a guide.

[0129] ■Experiment 4 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound IV (compound IV concentration: 0.075 mg / mL) was administered into the tail vein of these mice. One day later, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened. Near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo) (Figure 6, right: near-infrared fluorescence image).

[0130] As a comparative example, a saline solution containing compound IV was administered into the tail vein of normal mice (body weight approximately 20g). One day later, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and then laparotomy was performed. Near-infrared fluorescence imaging was then performed using a near-infrared fluorescence imaging device (pde-neo, Hamamatsu Photonics) (Figure 7).

[0131] Cancer tissue from bladder cancer model mice specifically exhibited near-infrared fluorescence (Figure 6, right: near-infrared fluorescence image), while normal tissue did not emit near-infrared fluorescence (Figure 7, right: near-infrared fluorescence image).

[0132] ■Experiment 5 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound IV (compound IV concentration: 0.075 mg / mL) was administered into the bladder of these mice via the urethra, and 10 minutes later, the bladder was irrigated five times with physiological saline via the urethra. Subsequently, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened, and near-infrared fluorescence observation was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo) (Figure 8, right: near-infrared fluorescence image).

[0133] As a comparative example, a saline solution containing compound IV was administered to the bladder of a normal mouse (body weight approximately 20g) via the urethra, and the bladder was flushed five times with saline via the urethra 10 minutes later. Subsequently, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened, and near-infrared fluorescence observation was performed using a near-infrared fluorescence imaging device (pde-neo, Hamamatsu Photonics, Ltd.) (Figure 9).

[0134] Cancer tissue from bladder cancer model mice specifically exhibited near-infrared fluorescence (Figure 8, right: near-infrared fluorescence image), while normal tissue did not emit near-infrared fluorescence (Figure 9, right: near-infrared fluorescence image).

[0135] ■Experiment 6 Two types of bladder cancer model mice (approximately 20g in weight) were used, in which MB49 mouse bladder cancer cells were transplanted into the bladder mucosa and grown for 3 or 4 weeks. 0.2 mL of physiological saline solution containing compound IV (compound IV concentration: 0.075 mg / mL) was administered into the tail vein of these mice. One day later, the mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg) and the abdomen was opened. Near-infrared fluorescence was observed using a near-infrared fluorescence imaging device (pde-neo, Hamamatsu Photonics), and the cancerous tissue area that emitted near-infrared fluorescence was removed and observed for near-infrared fluorescence (Figure 10, right: near-infrared fluorescence image).

[0136] Since cancer tissue fluoresces when irradiated with near-infrared light, according to the present invention, tumor tissue could be appropriately removed by using the fluorescence emitted by the cancer tissue as a guide.

[0137] ■Experiment 7 Based on the method reported by Okada et al., a mouse model of kidney cancer was created (Okada et al., Jpn. Arch. Int. Med., 1982, 29, 485). Specifically, rats (280g-320g) were fed after being continuously administered iron-nitrilotriacetate intraperitoneally for 6 months, and renal epithelial cell tumors were induced.

[0138] These rats were anesthetized by intraperitoneal administration of pentobarbital sodium salt, and a saline solution containing compound III was administered into the tail vein (compound dose: 0.75 mg / kg rat). At 30 minutes, 3 hours, and 24 hours after administration, the abdomen was opened, and near-infrared fluorescence imaging of the kidneys was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics PDE) (Figure 11). The kidneys that had been imaged with near-infrared fluorescence were also dissected from the body, and near-infrared fluorescence imaging of the cross-section was performed (Figure 12). Three rats were used at each time interval after administration, for a total of nine rats used.

[0139] Following continuous intraperitoneal administration of iron-nitrilotriacetate, macroscopic near-infrared fluorescence imaging of the surface of kidneys with tumors and pretumors revealed localized, strong fluorescence in the form of punctates. This fluorescence appeared to be located in the cortical region beneath the epidermis, rather than on the renal epidermis itself (magnified image at the bottom of Figure 11).

[0140] Furthermore, near-infrared fluorescence images of kidney cross-sections showed areas of strong, punctate fluorescence in the renal cortex, including the glomeruli and tubules (magnified image at the bottom of Figure 12), but no such punctate fluorescence was observed in the renal medulla (especially the inner zone of the renal medulla where tubules are absent).

[0141] The fluorescence pattern observed upon near-infrared light irradiation was not seen in normal kidneys and is specific to the kidneys of rats administered iron-nitrilotriacetate. Furthermore, near-infrared fluorescence was not observed in the kidneys of normal rats not administered compound III, nor in rats administered iron-nitrilotriacetate, at the same fluorescence measurement sensitivity. These test results reveal that in kidneys with tumors and pretumors treated with iron-nitrilotriacetate, areas of strong fluorescence intensity are present in the renal cortex.

[0142] Furthermore, after ethanol fixation, sectioning, and hematoxylin-eosin staining, specimens for microscopic observation were prepared and observed using bright-field and near-infrared fluorescence microscopy (Figure 13). Specifically, microscopic images of tissue sections from tumor kidneys treated with iron-nitrilotriacetate were obtained using a near-infrared fluorescence microscope, revealing locally strong near-infrared fluorescence in non-tumorous degenerative areas, pre-tumorous degenerative areas, and tumorous degenerative areas. Tumors embedded in the surrounding tissue were detectable. These macrofluorescence and microfluorescence images showed abnormal fluorescence patterns in pre-tumorous and tumorous areas.

[0143] ■Experiment 8 A mouse allogeneic tumor transplantation model was created as follows, based on "Cancer - Creation and Utilization of Disease Models" (Takuro Nakamura, L.I.C., 2012). (Breast cancer) A tumor model was created by subcutaneously administering mouse tumor cells 4T1 to normal Balb / c mice (body weight approximately 20g-30g) and allowing them to grow for 14 days. (Colon cancer) A tumor model was created by subcutaneously administering CT26 (colon cancer cells) to normal Balb / c mice (body weight approximately 20g-30g) and allowing them to grow for 14 days. (Skin cancer) B16F1 (skin cancer cells) were subcutaneously administered to C57BL / 6 mice (body weight approximately 15g-20g) and allowed to grow for 14 days to create a tumor model.

[0144] Next, 0.02-0.05 mL of physiological saline solution containing compound III or ICG (concentration of compound III or ICG: 1 mg / mL) was administered to the model mouse tumors, and transcutaneous fluorescence imaging was performed using a near-infrared fluorescence camera (Fluobeam, manufactured by Fluoptics, http: / / www.solve-net.com / info / wp-content / uploads / downloads / 2013 / 12 / Fluobeam.pdf) (excitation wavelength: 780 nm, detection wavelength: 800 nm or higher).

[0145] Fluorescence images for each tumor model are shown in Figures 14 to 17. Fluorescence imaging confirmed that compound III, administered intratumorally, remained within the tumor, did not leak fluorescence signals into the surrounding tissue, and enhanced the tumor's appearance (Figure 14: breast cancer, Figure 15: colorectal cancer, Figure 16: skin cancer).

[0146] Furthermore, when fluorescence imaging was performed during the visual tumor excision procedure, the tumors isolated from the mice and picked up with tweezers were clearly fluorescently enhanced, confirming that the administered compound did not extrude into the surrounding tissue and was able to visualize the tumor contour (Figure 17). In addition, some tumor tissue exhibiting a fluorescent signal remained in the mouse tissue, indicating the presence of residual tumor tissue that could not be completely removed by visual excision, suggesting the possibility of further excision of this tissue. On the other hand, ICG administration did not produce a fluorescent signal outside the tumor tissue, and therefore could not visualize the tumor contour.

[0147] ■Experiment 9 Under anesthesia induced by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), metastatic BALB / c mouse spontaneous renal cell carcinoma cell line RenCa cells (1 × 10⁶) were induced. 6 Compound III (purchased from cells, CLS Cell Lines Service GmbH, Eppelheim, Germany) was prepared in RPMI-1640 medium (fetal bovine serum-free, Fujifilm Wako Pure Chemical Industries) and inoculated into mice (BALB / cCrSlc, SPF, female, 7 weeks old) via tail vein administration. The mice were reared for 18 days to induce lung cancer. These mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (120 nmol / kg body weight) was administered via tail vein. Ten minutes later, the lungs were removed and near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo). It was found that the near-infrared fluorescence intensity of compound III was low in the lung cancer area (area enclosed in a frame in the near-infrared fluorescence image) (Figure 18).

[0148] The above results demonstrate that the location of lung cancer tissue can be identified by the area where the near-infrared fluorescence intensity of compound III is low.

[0149] ■Experiment 10 Under anesthesia induced by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), metastatic BALB / c mouse spontaneous renal cell carcinoma cell line RenCa cells (1 × 10⁶) were induced. 6 Cells (purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) was intravenously inoculated into mice (BALB / cCrSlc, SPF, female, 7 weeks old) and reared for 18 days to induce liver cancer. These mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (120 nmol / kg body weight) was administered via tail vein. Ten minutes later, the abdomen was opened and near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo). Near-infrared fluorescence of compound III from normal liver tissue was strong, while the emission of compound III from the liver cancer site (area enclosed in the frame in the right image) was remarkably weak, allowing for differentiation between normal and cancerous areas by fluorescence (Figure 19).

[0150] The above results demonstrate that the location of liver cancer tissue can be identified based on the fluorescence of compound III.

[0151] ■Experiment 11 Human gastric cancer cells MKN45 (1 × 10) 7Cells (purchased from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition) were subcutaneously transplanted into one side or the center of the back at the base of the forelegs of nude mice (BALB / cSlc-nu, SPF, male, 5 weeks old). These mice were reared for 10 days to create mice in which gastric cancer tissue was grown. These mice (body weight approximately 20g) were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (120 nmol / kg body weight) was administered via tail vein. Near-infrared fluorescence was observed from the upper skin on the back using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo). Near-infrared fluorescence of the cancerous tissue was stronger than that of normal tissue, allowing for differentiation between cancerous tissue (area enclosed by dotted lines) and normal tissue (Figure 20).

[0152] The above results demonstrate that gastric cancer tissue and normal tissue can be distinguished by the difference in near-infrared fluorescence intensity obtained by observing the near-infrared fluorescence of compound III.

[0153] ■Experiment 12 Metastatic BALB / c mouse spontaneous renal cell carcinoma line · RenCa cells (5 × 10⁻¹⁰) 6 Cells (purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) was inoculated intraperitoneally into mice (BALB / cCrSlc, SPF, female, 7 weeks old) and reared for 14 days to induce peritoneal cancer. These mice (approximately 20g body weight) were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (12 nmol / kg body weight) was administered via tail vein. Immediately after administration, the abdomen was opened and near-infrared fluorescence imaging was performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo). The near-infrared fluorescence of compound III from the peritoneal cancer site (area enclosed in a frame in the near-infrared fluorescence image) was stronger than that of normal tissue, and the peritoneal cancer site could be indicated by emission (Figure 21).

[0154] The above results demonstrate that the location of peritoneal cancer tissue can be identified based on the fluorescence of compound III.

[0155] ■Experiment 13 Human gastric cancer cells MKN45-Luc(5 × 10) 6 Nude mice (BALB / cSlc-nu, SPF, male, body weight approximately 20g) with peritoneal dissemination were generated by intraperitoneal injection of cells (purchased from the JCRB cell bank of the National Institute of Biomedical Innovation, Health and Nutrition) and rearing for 15 days. These mice (cancer mice) and normal nude mice (BALB / cSlc-nu, SPF, male, 5 weeks old) were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (40 nmol / kg body weight) was administered via tail vein. Near-infrared fluorescence of compound III was observed after administration using a near-infrared fluorescence imaging device (Hamamatsu Photonics pde-neo). No near-infrared fluorescence was emitted from the peritoneal cavity of normal mice (Figure 22). In cancer mice, the visible cancerous tissue enclosed by the dotted line in the right panel of Figure 23 was close to necrotic and did not emit near-infrared fluorescence, but the dissemination present throughout the abdominal cavity did emit near-infrared fluorescence (Figure 23).

[0156] The above results demonstrate that peritoneal dissemination originating from gastric cancer can be imaged by detecting the near-infrared fluorescence of compound III.

[0157] ■Experiment 14 Under anesthesia induced by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), RenCa renal cancer cells (5 × 10) were examined. 6 Cells (purchased from CLS Cell Lines Service GmbH, Eppelheim, Germany) were transplanted into the left kidney of mice (approximately 20g body weight) and reared for 9 to 14 days to create renal cancer mice. These mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (12 nmol / kg body weight) was administered via tail vein to the renal cancer mice. Ten minutes later, the near-infrared fluorescence of the kidneys was observed using a near-infrared fluorescence imaging system (pde-neo, Hamamatsu Photonics). Fluorescence from cancerous tissue was weak, while fluorescence from normal tissue was strong, allowing for differentiation between cancerous and normal tissue (Figure 24).

[0158] The above results demonstrate that it is possible to distinguish between renal cancer tissue and normal tissue by detecting the near-infrared fluorescence intensity of compound III.

[0159] ■Experiment 15 Human esophageal cancer cells KYSE850 were subcutaneously transplanted into the necks of nude mice (BALB / cSlc-nu, SPF, male, 5 weeks old) and reared for 18 days to create mice in which esophageal cancer was grown. The human esophageal cancer cells KYSE850 were purchased from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition (registration number: JCRB1422, established by: Hiroshi Shimada, Department of Nanobio-Medicinal Creation Science, Graduate School of Pharmaceutical Sciences, Kyoto University).

[0160] (Literature) Shimada Y. Imamura M, Wagata T, Yamaguchi N and Tobe T. Characterization of Twenty One Newly Established Esophageal Cancer Cell Lines. Cancer, 69: 277-284 (1992). Tanaka H, ​​Shibagaki I, Shimada Y, Wagata T, Imamura M, Ishizaki K. Characterization of p53 gene mutations in esophageal squamous cell carcinoma cell lines: increased frequency and different spectrum of mutations from primary tumors. Int J Cancer. 65:372-376 (1996). Tanaka H, ​​Shimada Y, Imamura M, Shibagaki I, Ishizaki K. Multiple types of aberrations in the p16 (INK4a) and the p15(INK4b) genes in 30 esophageal squamous-cell-carcinoma cell lines. Int J Cancer. 70:437-442 (1997).

[0161] Next, mice were anesthetized by subcutaneous administration of ketamine (75 mg / kg) and medetomidine (1 mg / kg), and then compound III (120 nmol / kg body weight) was administered via tail vein. Near-infrared fluorescence imaging of compound III was then performed using a near-infrared fluorescence imaging device (Hamamatsu Photonics PDE). Near-infrared fluorescence was strong from cancerous tissue (areas enclosed in circular frames) and weak from normal tissue (Figure 25). The fluorescence within the oval frames represents near-infrared fluorescence from the left and right kidneys.

[0162] The above results demonstrate that esophageal cancer tissue and normal tissue can be distinguished by detecting the near-infrared fluorescence of compound III.

[0163] ■Experiment 16 A mouse model of human cancer cell transplantation was created as follows. (Head and neck / tongue cancer) Immunodeficient SCID mice (body weight 17g-23g) were given approximately 10 human tongue cancer cells CAL27 (purchased from American Type Culture Collection: ATCC). 6 A tumor model was created by subcutaneously administering the cells and allowing them to grow for 49 days. (Brain Tumor / Neuroblastoma) Immunodeficient SCID mice (body weight 17g-23g) were given approximately 10 human neuroblastoma cells SH-SY5Y (purchased from ATCC). 6 A tumor model was created by subcutaneously administering the cells and allowing them to grow for 90 days. (Brain tumor, glioblastoma) Approximately 10 human glioblastoma cells U-251MG (obtained from the European Collection of Authenticated Cell Cultures) were subcutaneously administered to immunodeficient SCID mice (body weight 17 g to 23 g) and grown for 90 days to create a tumor model. 6 After that, 0.02 to 0.1 mL of an aqueous physiological saline solution containing Compound III (concentration of Compound III: 1 mg / mL) was topically administered to the model mouse tumors, and transcutaneous fluorescence imaging was performed using a near-infrared fluorescence camera (Fluobeam manufactured by Fluoptics) (excitation wavelength: 780 nm, detection wavelength: 800 nm or more).

[0164]

[0165] Fluorescence images immediately after administration or 50 minutes to 1 hour after administration in each tumor model are shown in FIGS. 26 to 28. It was confirmed that Compound III administered into the tumor remained inside the tumor by fluorescence imaging without leakage of the fluorescence signal to the periphery of the tumor, and contrasted the tumor appearance (FIG. 26: tongue cancer, FIG. 27: neuroblastoma, FIG. 28: glioblastoma).

Industrial Applicability

[0166] By administering a cyclodextrin-bound indocyanine compound represented by (Formula A) or a pharmaceutically acceptable salt thereof and observing the near-infrared fluorescence emitted by near-infrared light irradiation, cancer tissues and normal tissues of cancers such as bladder cancer, kidney cancer, gastric cancer, liver cancer, lung cancer, peritoneal seeding, peritoneal cancer, esophageal cancer, breast cancer, colorectal cancer, skin cancer, head and neck cancer, brain tumor, etc. can be discriminated, and the exact site of cancer tissue during cancer surgery can be identified. Therefore, the cyclodextrin-bound indocyanine compound represented by (Formula A) or a pharmaceutically acceptable salt thereof is useful as a cancer contrast agent.​

Claims

1. A composition for contrast imaging of cancer selected from the group consisting of bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, peritoneal cancer, peritoneal dissemination, esophageal cancer, breast cancer, colorectal cancer, skin cancer, head and neck cancer, and brain tumors, Below formula: 【Chemistry 1】 The composition comprising a cyclodextrin-bound indocyanine compound represented by or a pharmaceutically acceptable salt thereof.

2. The cyclodextrin-bound indocyanine compound is given by the following formula: 【Chemistry 2】 The composition according to claim 1, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

3. The cyclodextrin-bound indocyanine compound is given by the following formula: 【Transformation 3】 The composition according to claim 1, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

4. A liquid composition according to any one of claims 1 to 3.

5. The composition according to any one of claims 1 to 4, for contrast imaging of bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, peritoneal cancer, peritoneal dissemination, or esophageal cancer.

6. A composition according to any one of claims 1 to 4, for use as contrast for breast cancer, colorectal cancer, skin cancer, head and neck cancer, or brain tumors.

7. The composition according to any one of claims 5, for systemic administration.

8. The composition according to any one of claims 6, for local administration.

9. A composition according to any one of claims 1 to 3, for use as contrast imaging for bladder cancer and for intravesical administration via the urethra.

10. The composition according to claim 2, for contrast imaging of bladder cancer, gastric cancer, peritoneal cancer, peritoneal dissemination, or esophageal cancer, and used by systemic administration.

11. The composition according to claim 2, for contrast imaging of kidney cancer, lung cancer, or liver cancer, and for use by systemic administration.

12. The composition according to claim 2, for contrast imaging of breast cancer, colorectal cancer, skin cancer, head and neck cancer, or brain tumor, and used by local administration.

13. The composition according to claim 2, which is for contrast imaging of breast cancer and is used by local administration.

14. Use of a cyclodextrin-conjugated indocyanine compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 for preparing a contrast-enhancing composition for cancer selected from the group consisting of bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, peritoneal cancer, peritoneal dissemination, esophageal cancer, breast cancer, colorectal cancer, skin cancer, head and neck cancer, and brain tumors.

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