Novel agent for measuring xct functional activity, novel method for examining xct functional activity, novel radioactive diagnostic agent for cancer, and novel method for examining cancer

Abstract

To provide a means for measuring xCT functional activity.SOLUTION: The accumulation of 125I (R) - HPG in cancer cells is examined by using colon cancer cells, and the accumulation ratio of 125I (R) - HPG by xCT is found to be high by paying attention to xCT of a cancer-related amino-acid transporter.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an xCT functional activity measuring agent, a method for testing xCT functional activity, a cancer radioactive diagnostic agent, a method for testing cancer, iodine-labeled 4-hydroxyphenylglycine, a method for screening an xCT functional activity inhibitor, a method for screening an xCT functional activity promoter, an xCT functional activity measuring device, and a program for measuring xCT functional activity. [Background technology]

[0002] Cancer is the leading cause of death among Japanese people, killing more than 300,000 people annually. Cancer is diagnosed through imaging tests such as PET tumor scans, X-ray scans, ultrasound scans, and CT scans. PET tumor scans are the most common. 18 F-2-fluoro-2-deoxy-D-glucose ( 18 F-FDG) is used as a radioactive tracer (Non-Patent Documents 1 and 2). however, 18 F-FDG accumulates physiologically at high levels in normal tissues such as the brain and heart, which are active in energy metabolism, and in excretory tissues such as the kidneys, ureters, and bladder, making it difficult to image tumors in or around these tissues.

[0003] Cancer treatments include surgery, chemotherapy, and radiotherapy, and chemotherapy using anticancer drugs targeting xCT, a cancer-related amino acid transporter highly expressed in cancer cells, has recently attracted attention. xCT is involved in redox regulation, reducing reactive oxygen species (ROS) and promoting cancer cell survival by incorporating cystine to generate glutathione (GSH). Non-patent document 3 shows that when cystine uptake is inhibited using an xCT inhibitor or the like, the production of GSH is suppressed and the reduction of ROS is also inhibited, which changes redox regulation, and the redox regulation increases ROS, causing cancer cells to die.

[0004] The anti-inflammatory drug sulfasalazine inhibits the uptake of cystine by xCT, altering the activity of xCT and inducing anti-cancer effects. Its clinical application is being promoted as a novel anti-cancer drug with high affinity for xCT (Non-patent Document 4).

[0005] Patent Document 1 discloses the use of a radiotherapeutic or radiodiagnostic agent in the treatment or diagnosis of cell proliferative disorders. Patent Document 2 discloses the use of radioisotope-labeled amino acids in the diagnosis of bacterial infections. Patent Document 3 discloses a method for classifying bacteria or cells using radioisotope-labeled amino acids instead of radioactively labeled compounds, a method for classifying the transport properties of amino acids, a method for assisting in the diagnosis of bacterial infections, and a method for assisting in the diagnosis of cancer. Patent Document 4 discloses the use of radiolabeled compounds or radioisotope-labeled amino acids in bacterial infections. The above documents do not disclose or suggest the "agent for measuring xCT functional activity" of the present invention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6474547 [Patent Document 2] Patent Publication No. 2019-137686 [Patent Document 3] Patent Publication No. 2021-94023 [Patent Document 4] Patent Publication No. 2021-95401 [Non-patent literature]

[0007] [Non-Patent Document 1] van Oosten M, Hahn M, Crane LM: Targeted imaging of bacterial infections: advances, hurdles and hopes. FEMS Microbiol Rev,39(6):892-916(2015). [Non-patent document 2] LoveC, Tomas MB, Tronco CG, PalestroCJ:FDG PET of infection and inflammation.RadioGraphics, 25(5): 1375-1368(2005). [Non-patent document 3] Scott J. Dixon, Kathryn M. Lemberg, MichaelR. Lamprecht, et al. Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. cell. 149(5): 1060-1072; 2012. [Non-patent document 4] Kohei Otsubo, Kaname Nosaki, Chiyo K.Imamura, et al. Phase I study of salazosulfapyridine in combination with cisplatin and pemetrexed for advanced non-small-cell lung cancer. Cancer Sci.108(9):1843-1849;2017. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a means for measuring the functional activity of xCT. [Means for solving the problem]

[0009] As a result of extensive investigation, the present inventors have found that 125We investigated the accumulation of I-HPG in cancer cells and focused on xCT, a cancer-related amino acid transporter. 125 The present invention was completed based on the discovery that I-HPG has a high accumulation rate.

[0010] That is, the present invention is as follows. 1. 125 An agent for measuring xCT functional activity, comprising l-4-hydroxyphenylglycine. 2. A method for testing or assisting in testing xCT functional activity, comprising the steps of: (1) administering the xCT functional activity measuring agent described in the preceding paragraph 1 to a subject or a test sample; (2) detecting the administered xCT functional activity measuring agent; (3) determining the xCT functional activity from the detection results; 3. 125 A cancer radioactive diagnostic agent containing I-4-hydroxyphenylglycine. 4. A cancer testing method or testing assistance method, comprising the following steps: (1') administering the radioactive diagnostic agent according to the preceding paragraph 3 to a subject or a test sample; (2') detecting the administered radioactive diagnostic agent; (3') A step of determining the presence or absence of cancer based on the detection results. 5. 125 A kit for measuring xCT functional activity and / or diagnosing cancer, comprising l-4-hydroxyphenylglycine. 6.xCT 125 I- A method for testing xCT functional activity, comprising the step of measuring the rate of accumulation of 4-hydroxyphenylglycine in cancer cells. 7. The method for testing xCT functional activity according to the preceding paragraph 6, further comprising the following steps: (A) administering the xCT functional activity measuring agent described in the preceding paragraph 1 to a test sample in the absence of Na+; (B)Na + into cancer cells in the absence of 125 The accumulation rate of I-4-hydroxyphenylglycine was +In the absence of System Xc-inhibitors (especially sulfasalazine (SSZ) or cystine (Cys-Cys)), the tumor cells were treated with 125 A process of calculating the contribution rate of System Xc-, which is the value obtained by subtracting the accumulation rate of I-HPG. 8. Represented by the following formula (II): 125 I-4-hydroxyphenylglycine. [ka] 9. A method for screening for an xCT functional activity inhibitor or an xCT functional activity promoter, comprising the steps of: (1) 125 administering an xCT functional activity measuring agent containing l-4-hydroxyphenylglycine and a test compound to a test sample; (2) detecting the administered xCT functional activity measuring agent; (3) A step of determining, based on the detection results, a test compound that reduces the xCT functional activity as an xCT functional activity inhibitor, or a test compound that improves the xCT functional activity as an xCT functional activity promoter. 10. An xCT functional activity measuring device, comprising the following means: 1) Included in the test sample 125 A method for measuring the accumulation of I-4-hydroxyphenylglycine 2) Measured by the method 125 A method for calculating the contribution rate of System Xc-, an index of xCT functional activity, from the accumulation of l-4-hydroxyphenylglycine 11. A program for measuring xCT functional activity, Computer 125 I- A program that functions as a means of calculating the contribution rate of System Xc-, an indicator of xCT functional activity, from the amount of 4-hydroxyphenylglycine accumulated. [Effects of the Invention]

[0011] The present invention is capable of measuring the functional activity of xCT. [Brief explanation of the drawings]

[0012] [Figure 1] Labeling ratio of 125I-HPG (Example 1) [Figure 2] Chromatogram of 125I-HPG analyzed by HPLC (Example 1). [Figure 3] Gene expression levels of amino acid transporters in each cancer cell line (Example 2). [Figure 4] Accumulation amount due to differences in xCT gene expression level (Example 2) [Figure 5] Method for evaluating the contribution of the amino acid transport system (Example 2) [Figure 6] 1 shows the accumulation rate of 125I-HPG in LS180 cells after loading with a specific inhibitor of amino acid transporters (Example 2). [Figure 7] 1 shows the accumulation rate of 125I-HPG in DLD-1 cells after loading with a specific inhibitor of amino acid transporters (Example 2). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to the cancer-related amino acid transporter xCT. 125 The present invention has been achieved by newly discovering the relationship between I-HPG and the IL-1 receptor.

[0014] (xCT functional activity measuring agent) The present invention provides 125 The xCT functional activity measuring agent of the present invention comprises I-HPG. 125 By accumulating I-HPG in a subject or test sample (especially within cancer cells), the function of xCT can be visualized. Visualizing this function not only enables cancer imaging diagnosis but also imaging of intracellular redox regulation. Here, "redox regulation" refers to the control of protein activity by changing the redox state of the protein (cysteine ​​thiol group). Furthermore, visualizing the function of xCT can provide important diagnostic information related to the effectiveness of treatments, including curative drug therapies targeting cancer cells. Furthermore, in cancer cells after X-ray irradiation, even though the expression level of the xCT gene is reduced, xCT per transporter in xCT may be activated. In such cases, it is possible to measure the functional activity of xCT after X-ray irradiation, which cannot be predicted from the expression level of the xCT gene.

[0015] ( 125 I-HPG) The xCT functional activity measuring agent of the present invention is 125 I-HPG" is 125-iodine ( 125 I) labeled hydroxyphenylglycine. 125 I-HPG can be produced and purified by any method known per se or developed in the future. For example, commercially available HPG can be directly labeled using chloramin-T as an oxidizing agent. 125 Labeled with I, 125 I-HPG can be obtained. Furthermore, obtained 125 I-HPG can be purified using high performance liquid chromatography known per se.

[0016] 125 I-HPG can be represented, for example, by the following formula (II): [ka]

[0017] 125 More specifically, I-HPG can be represented by, for example, the following formula (I). [ka]

[0018] (xCT) In this invention, xCT stands for system X - c(system X -Amino acid transporters are membrane proteins that transport amino acids, which are hydrophilic compounds, through the cell membrane and consist of a variety of molecular species. + Transports substrates using the concentration gradient of substances other than Na + Independent amino acid transporters and Na + Using the concentration gradient of Na + There are also other amino acid transporters that are dependent on the ATP. These amino acid transporters are involved in system X - c, system PAT, system L, system asc, system y + , system T, system b 0,+ , system y + It is classified as an amino acid transport system such as L. "xCT" is an amino acid transporter that exchanges cystine and glutamate. It is expressed in normal tissues such as the brain and macrophages, but is a "cancer-associated amino acid transporter" whose expression has been confirmed to be elevated in cancer cells. When xCT increases the uptake of cystine from outside the cell, the increased cystine promotes the production of the antioxidant glutathione (GSH). This increases the resistance of cancer cells to oxidative stress, allowing them to survive (redox control), so measuring the functional activity of xCT is important. Here, "function of xCT" means that xCT 125 It refers to the uptake of I-HPG, cystine, etc. into cancer cells. The "functional activity of xCT in cells (cancer cells)" can be defined as follows, taking into account the above explanation and the examples below, but is not particularly limited to these. The functional activity of xCT in cells (cancer cells) is about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2.0-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3.0-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, or about 5.0-fold higher per cell (or per transporter) than that of normal cells or normal cells after X-ray irradiation (cancer cells after X-ray irradiation). 125 This means that I-HPG and cystine are accumulating. In this specification, when a numerical range (e.g., accumulation amount) is described in stages, the respective lower and upper limits can be independently combined. For example, "about 1.6 times" and "about 2.5 times" can be combined to form "about 1.6 times to about 2.5 times."

[0019] (Method for testing xCT functional activity or method for assisting testing) The present invention also encompasses a method for testing or a method for assisting in testing xCT functional activity, which comprises the following steps: (1) A step of administering the xCT functional activity measuring agent of the present invention to a subject or a test sample. (2) detecting the administered xCT functional activity measuring agent; (3) determining the xCT functional activity from the detection results;

[0020] Regarding (1), the method of administering the xCT functional activity measuring agent of the present invention to a subject is not particularly limited, and examples include intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection. The timing of administration can be appropriately determined depending on the condition of the subject (subject) and the circumstances of treatment and diagnosis. The "test sample" to be subjected to the testing method of the present invention may be a specimen obtained from a living body itself, or may be prepared from a specimen by any method known per se or to be developed in the future. Examples of such samples include non-liquid samples such as cancer tissues and cancer cells, and liquid samples such as blood and saliva. The cancer tissues may be frozen tissues obtained by freezing after collection from a subject, or pathological tissues obtained by histopathological processing. Examples of such pathological tissues include formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues.

[0021] Regarding (2), the step of detecting the xCT functional activity measuring agent administered to the subject is not particularly limited, and can be performed, for example, by imaging using a known method. For example, xCT function (cancer cells or cancer tissues in the patient's body) can be imaged by detecting radiation emitted from the compound using nuclear medicine imaging such as PET (Positron Emission Tomography) or SPECT (Single Photon Emission Computed Tomography). The step of detecting the xCT functional activity measuring agent administered to the test sample is not particularly limited, and can be performed using, for example, a gamma counter.

[0022] The present invention 125 The radioactivity of I-HPG can be appropriately set to the detectable amount in step (2). 123 I, etc., if it is PET imaging 18 It can also be combined with a radiolabeled substance such as F. For example, in the case of PET imaging, the radioactivity must be such that PET imaging is possible. For example, for the purpose of performing PET imaging on an adult, it is sufficient to have a radioactivity of 50 to 225 MBq at the time of use.

[0023] Regarding (3), the method for determining the xCT functional activity from the detection results is not particularly limited. For example, the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample can be determined by the method of the present invention. 125The signal intensity and / or signal distribution of radioactivity detected in the same site or in a cancer-negative sample of a control subject known to be cancer-free (cancer-negative control subject) administered I-HPG, or 125 This can be determined by comparing the signal intensity and / or signal distribution of radioactivity detected in the same area or in a cancer-positive sample from a control subject known to have cancer (cancer-positive control subject) who was administered I-HPG. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample is equal to or less than the signal intensity and / or signal distribution of radioactivity detected in the same area of ​​a cancer-negative comparison subject or cancer-negative sample, it can be determined that xCT functional activity has decreased. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample is higher than the signal intensity and / or signal distribution of radioactivity detected in the same area of ​​a cancer-negative comparison subject or cancer-negative sample, the xCT functional activity can be determined to be high. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample is equal to or greater than the signal intensity and / or signal distribution of radioactivity detected in the same area of ​​a cancer-positive comparison subject or cancer-positive sample, the xCT functional activity can be determined to be high. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample is lower than the signal intensity and / or signal distribution of radioactivity detected in the same area of ​​a cancer-positive comparison subject or cancer-positive sample, the xCT functional activity can be determined to be low.

[0024] Furthermore, the xCT functional activity after X-ray irradiation can also be measured in the test method of the present invention. 125 The signal intensity and / or signal distribution of I-HPG can be determined by comparing the same site of the subject or test sample before and after X-ray irradiation. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample after X-ray irradiation is the same as or increased from the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample before X-ray irradiation, it can be determined that xCT functional activity has increased. If xCT functional activity has been determined to have increased, it can also be determined that xCT per transporter in xCT is active or that cancer cells are activated. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample after X-ray irradiation is less than the same as the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample before X-ray irradiation, it can be determined that xCT functional activity has decreased.

[0025] Furthermore, the testing method of the present invention can also be used to assess the therapeutic effect of an xCT inhibitor. By visualizing the function of xCT, the testing method of the present invention enables not only cancer imaging diagnosis but also imaging of intracellular redox regulation, making it possible to assess the therapeutic effect of an xCT inhibitor in particular. Furthermore, the testing method of the present invention is expected to be used not only to assess the therapeutic effect of existing xCT inhibitors but also that of xCT inhibitors in development. Specifically, in the above (3), the determination can be made, for example, by comparing the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample before and after treatment with an xCT inhibitor. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample before treatment is higher than the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample after treatment, it can be determined that the anti-cancer therapeutic effect of the xCT inhibitor is high. For example, if the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample before treatment is lower than the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample after treatment, it can be determined that the cancer treatment effect of the xCT inhibitor is low.

[0026] As used herein, the term "xCT inhibitor" includes any xCT inhibitor that is known per se or that will be developed in the future, such as sulfasalazine, erastin, sorafenib, anti-xCT antibodies, and the like. As used herein, "pre-treatment" refers to the period before administration of an xCT inhibitor or cancer treatment. Specifically, it includes the period before starting administration of an xCT inhibitor or a cancer therapeutic agent. "Post-treatment" refers to the period after starting treatment with an xCT inhibitor or a cancer therapeutic agent, during which the therapeutic effect of the xCT inhibitor or the cancer therapeutic agent is confirmed after a certain period of time.

[0027] The examination method of the present invention is 125 The functional activity of xCT can also be measured by measuring the rate of accumulation of I-HPG in cancer cells. Specifically, the following can be included: (A)Na + a step of administering the xCT functional activity measuring agent of the present invention to a test sample in the absence of (B)Na + into cancer cells in the absence of 125 From the accumulation rate of I-HPG, Na + In the absence of System Xc-inhibitors (especially sulfasalazine (SSZ) or cystine (Cys-Cys)), the tumor cells were treated with 125 A step of subtracting the accumulation rate of I-HPG.

[0028] (B) is explained using Figure 5. + into cancer cells in the presence 125 The accumulation rate of I-HPG was defined as 100%. The difference in the inhibitory effect when MeAIB ("Na MeAIB" and "Ch MeAIB" in Figure 5) was added in the presence (Na) and absence (Ch) of Na was defined as the contribution rate of System A. The inhibitory effect when BCH ("Ch BCH" in Figure 5) was added in the absence of Na was defined as the contribution rate of System L. The inhibitory effect when SSZ ("Ch SSZ" in Figure 5) or Cys-Cys ("Ch Cys-Cys" in Figure 5) was added was defined as the contribution rate of System Xc. Furthermore, for the calculated xCT125 From the cancer cell accumulation rate of I-HPG, after xCT correction 125 The rate of I-HPG accumulation in cancer cells can be measured. Examples of inhibitors for system PAT include α-(methylamino)isobutyric acid (MeAIB), inhibitors for system L include 2-amino-2-norbonanecarboxylic acid (BCH), and inhibitors for system Xc- include Cys-Cys, SSZ, erastin, and sorafenib.

[0029] (Cancer diagnosis, cancer testing methods, or testing assistance methods) 125 I-HPG can also be used for cancer diagnosis and testing. 125 This also includes cancer radioactive diagnostic agents containing I-HPG, cancer testing methods or testing auxiliary methods, etc. A cancer testing method or testing assistance method, characterized by including the following: (1') A step of administering the radioactive diagnostic agent of the present invention to a subject or a test sample. (2') detecting the administered radioactive diagnostic agent (3') A step of determining the presence or absence of cancer from the detection results

[0030] For (1') and (2'), please refer to (1) and (2) above. In (3'), the method for determining the presence or absence of cancer from the detection results is not particularly limited, but for example, the signal intensity and / or signal distribution of radioactivity detected in a subject or test sample can be determined by the method of the present invention. 125 The signal intensity and / or signal distribution of radioactivity detected in the same site or in a cancer-negative sample of a control subject known to be cancer-free (cancer-negative control subject) administered I-HPG, or 125This can be determined by comparing the signal intensity and / or signal distribution of radioactivity detected in the same area or in a cancer-positive sample from a control subject known to have cancer (cancer-positive control subject) who was administered I-HPG.

[0031] (cancer) 125 When I-HPG is used for cancer diagnosis or testing, the target cancer is not particularly limited and may be a solid cancer or a blood cancer, such as colorectal cancer (rectal cancer or colon cancer), liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, adenocarcinoma, skin cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, kidney cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, small intestine cancer, gallbladder cancer, biliary tract cancer, testicular cancer, ovarian cancer, breast cancer, leukemia, malignant lymphoma, osteosarcoma, multiple myeloma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, and soft tissue sarcoma.

[0032] Pharmaceutically Acceptable Carriers The xCT functional activity measuring agent or cancer radioactive diagnostic agent of the present invention comprises: 125 In addition to I-HPG, the formulation may contain a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid excipient, diluent, lubricant, or solvent for encapsulating a substance. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject to which it is administered. Specific examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, sucrose, etc.; starches such as corn starch and potato starch; celluloses such as cellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, etc.; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; physiological saline; Ringer's solution; ethyl alcohol; polyanhydrides such as polyester and polycarbonate.Those skilled in the art can appropriately select these carriers.

[0033] (Dosage form) The dosage form of the xCT functional activity measuring agent or cancer radiodiagnostic agent of the present invention is not particularly limited, and may be administered parenterally as, for example, an injection, suppository, transdermal agent, inhalant, etc., or orally as a powder, granules, tablet, capsule, pill, liquid, etc. Those skilled in the art can appropriately select and prepare such dosage forms using methods known per se.

[0034] (kit) The present invention provides 125 The kit of the present invention may also include a kit for measuring xCT functional activity and / or diagnosing cancer, which contains I-HPG. The kit of the present invention may also include instructions for administering each active ingredient.

[0035] (Method for screening xCT functional activity inhibitors and xCT functional activity promoters) The present invention also relates to a method for screening for an xCT functional activity inhibitor and an xCT functional activity promoter, which comprises the following steps: (1) 125a step of administering an xCT functional activity measuring agent containing l-4-hydroxyphenylglycine and a test compound to a test sample; (2) detecting the administered xCT functional activity measuring agent; (3) determining, based on the detection results, that a test compound that reduces xCT functional activity is an xCT functional activity inhibitor, or that a test compound that improves xCT functional activity is an xCT functional activity promoter. The above-described methods can be appropriately adopted for the "step of detecting an agent for measuring xCT functional activity" in (2) above. Any substance can be used as a "test compound" in the present invention. The type of test compound is not particularly limited, and may be a known therapeutic agent, an individual low-molecular-weight synthetic compound (particularly siRNA), a compound present in a natural product extract, or a synthetic peptide. Alternatively, the test compound may be a compound library, a phage display library, or a combinatorial library. The test compound is preferably a low-molecular-weight compound, and a compound library of low-molecular-weight compounds is preferred. Construction of a compound library is known to those skilled in the art, and commercially available compound libraries can also be used. The determination method (3) can be achieved by comparing the xCT functional activity value of a test sample containing a test compound with that of a control sample not containing the test compound. For example, if the xCT functional activity value of a test sample containing a test compound is higher than that of a test sample not containing the test compound, the test compound can be determined to be an xCT functional activity promoter.

[0036] (xCT functional activity measurement program) The present invention relates to a computer 125 I- This also includes programs that function as a means of calculating the contribution rate of System Xc-, an indicator of xCT functional activity, from the amount of 4-hydroxyphenylglycine accumulated. This program is stored, for example, on a hard disk drive, and when executed, the necessary programs and data are loaded into RAM. The loaded program is executed by the CPU, thereby realizing various processing contents on the computer.

[0037] (xCT functional activity measuring device) The present invention also relates to an xCT functional activity measuring device comprising the following means: 1) Included in the test sample 125 A method for measuring the accumulation of I-4-hydroxyphenylglycine 2) Measured by the method 125 A method for calculating the contribution rate of System Xc-, an index of xCT functional activity, from the accumulation of l-4-hydroxyphenylglycine The means for measuring the accumulation amount in 1) above is not particularly limited, but examples thereof include known liquid chromatography mass spectrometers, nuclear magnetic resonance spectrometers, electrophoresis devices, and ultraviolet spectrometers. The calculation means for the above 2) is the "Na + into cancer cells in the absence of 125 From the accumulation rate of I-HPG, Na + In the absence of System Xc-inhibitors (especially sulfasalazine (SSZ) or cystine (Cys-Cys)), the tumor cells were treated with 125 A means for subtracting the accumulation rate of I-HPG can be employed, and the above-mentioned program for measuring xCT functional activity can be built into the device.

[0038] The present invention will be specifically illustrated below with reference to examples, but the present invention is not limited to these examples. [Example]

[0039] (Labeling and purification of HPG) In this embodiment, 125 I was labeled with 4-hydroxyphenylglycine (HPG) and purified.

[0040] (labeling method) The labeling material was commercially available HPG, and radioactive iodine labeling was performed by the direct labeling method using chloramin-T (Nacalai tesque) as an oxidizing agent. HPG was dissolved in 0.1 N NaOH (Nacalais tesque):0.5 M phosphate buffer (Fujifilm Wako Pure Chemical Industries) = 7:3 phosphate buffer to prepare a 0.5 mM HPG solution. 125 The mixture was mixed with I-NaI (3.7 MBq, PerkinElmer), and 200 μL of chloramine T solution (0.3 mM in 0.5 M phosphate buffer) was added. The mixture was allowed to react at room temperature for 20 minutes. 15 minutes after the start of the reaction, 40 μL of a 1 / 10 saturated solution of sodium pyrosulfite (Fujifilm Wako Pure Chemical Industries) was added to stop the reaction. 125 The labeling efficiency of I-HPG was evaluated by thin-layer chromatography (TLC) analysis using silica gel thin-layer plates (silicagel 60F254, Merck) and radioactivity measurement using an Autowell γ counter (AccuFLEX γ7000, Aloka Medical). The developing solvent was ethanol:water:acetic acid = 5:44:1. 125 I-HPG was purified by high performance liquid chromatography (HPLC) using a 5C18 MS-2 Packed column (4.6 mm I.D. × 250 mm) at a flow rate of 0.7 mL / min. After separation and purification, radiochemical purity was determined by TLC analysis under the above conditions.

[0041] (result) The results of TLC analysis of the labeling reaction solution are shown in FIGS. 125 The labeling rate of I-HPG was 75% by TLC (Figure 1). By HPLC, the peak appearing between 6 and 7 minutes was collected. The target labeled compound was separated by HPLC and analyzed by TLC. 125 The radiochemical purity of I-HPG was 98% or higher (Figure 2), and stability was confirmed for 7 days. From the above results, by the chloramine T method, 125 I-HPG was obtained in a short time with high labeling efficiency and radiochemical purity. [Example]

[0042] (Specific inhibitory load of amino acid transport system 125 I- HPG accumulation experiment) System PAT (PAT1, PAT2, etc.), system L (LAT1, LAT2, LAT3, LAT4, etc.), system asc (asc1, etc.), system y+ (CAT1, CAT2, CAT3, CAT4, etc.), system Xc (xCT, etc.), system T (TAT1, etc.), system b0, + (BAT1, etc.), and system y+L (y+LAT1, y+LAT2, etc.) are Na+-independent amino acid transport systems that transport substrates using the concentration gradient of substances other than Na+. In this example, we used a specific inhibitor of the Na+-independent amino acid transport system to investigate the effects of Na+-independent amino acid transport on the human colon cancer cell lines (LS180, DLD-1). 125 I-HPG accumulation was evaluated. In this and subsequent examples, LS180 cells were cultured in E-MEM medium at 1 × 10 cells per well the day before. 5 The cells were seeded at a cell number adjusted to 1 × 10 per well. DLD-1 cells were seeded in D-MEM medium the day before. 5 The cells were seeded after adjusting the number of cells to the desired number. In this and subsequent examples, the purified product in Example 1 125 I-HPG was used.

[0043] (2-1) Measurement of amino acid transporter gene expression levels by quantitative PCR The expression levels of the xCT gene in LS180 and DLD-1 were evaluated by absolute quantitative analysis using real-time polymerase chain reaction (PCR). Table 1 shows the primer sequences and final concentrations of the xCT gene used in real-time PCR. RNA was extracted from LS180 and DLD-1, cDNA was synthesized, and the expression levels of the xCT gene in LS180 and DLD-1 were measured by real-time PCR.

[0044] [Table 1]

[0045] (2-2) 125 Cell accumulation experiment using I-HPG For LS180 and DLD-1 125 I-HPG 18.5 kBq / well was administered. 125 Five minutes after administration of I-HPG, the cell surface of each cancer cell and the well were washed twice with Ch-PBS, dissolved in 500 μL of 0.1 N NaOH aqueous solution, and the amount of I-HPG administered to the cancer cells was measured using a gamma counter. 125 The accumulation of I-HPG was measured. After the measurement, the amount of cellular protein in each well was measured using a Multiskan FC absorbance microplate reader (Thermo Scientific). 125 The accumulation amount of I-HPG was corrected.

[0046] (result) The expression level of the xCT gene in each cancer cell line determined by real-time PCR is shown in Figure 3. When comparing the xCT gene expression levels between LS180 and DLD-1, the xCT gene expression level in LS180 was higher than that in DLD-1. In the presence of sodium, without inhibitor loading 125 The results of I-HPG accumulation are shown in Figure 4. When comparing the xCT gene expression levels in LS180 and DLD-1 together with the results in Figure 3, it was confirmed that LS180 had a higher level of xCT gene expression, and that the accumulation level in LS180 was correspondingly higher than that in DLD-1.

[0047] (2-3) Specific inhibitory load on amino acid transport system 125 I-HPG integration experiment In the loading of specific inhibitors of amino acid transport systems 125 The I-HPG accumulation experiment was performed based on the method of Shikano et al. (Shikano N et al., Ann Nucl Med. 2004 18(3):227-34). Based on the xCT gene expression level determined by real-time PCR, two human colon cancer cell lines created by the present inventors were selected: LS180, a cell line with high xCT gene expression, and DLD-1, a cell line with low xCT gene expression. 125 The contribution of xCT was examined based on its effect on I-HPG accumulation. The method for evaluating the contribution rate of amino acid transport systems used in this example is explained using Figure 5. The uptake of the control in the presence of Na without any inhibitor was defined as 100%. The difference in inhibitory effect when MeAIB ("Na MeAIB" and "Ch MeAIB" in Figure 5) was added in the presence (Na) and absence (Ch) of Na was defined as the contribution rate of System A. The inhibitory effect when BCH ("Ch BCH" in Figure 5) was added in the absence of Na was defined as the contribution rate of System L. The inhibitory effect when SSZ ("Ch SSZ" in Figure 5) or Cys-Cys ("Ch Cys-Cys" in Figure 5) was added was defined as the contribution rate of System Xc. The inhibitor MeAIB (Sigma-Aldrich) is α-(methylamino)isobutyric acid, which specifically inhibits system PAT. The inhibitor BCH (Sigma-Aldrich) is a 2-amino-2-norbonanecarboxylic acid that specifically inhibits system L. The inhibitor SSZ used was S0883-10G from Sigma-Aldrich. The inhibitor Cys-Cys used was 10328-04 from Nacalai Tesque. Because no specific inhibitors have been found for system Xc, system T, system b0, +, or system y+L, direct calculation of the xCT accumulation rate due to specific inhibitor loading is difficult. However, since system Xc is the only cancer-related amino acid transport system among these transport systems, the xCT transport fraction was evaluated by subtracting the inhibitory effect of SSZ or Cys-Cys from the Na+-independent accumulation in cancer cells (Figure 5). The buffer solution used was Na+-PBS or Na-free Ch (choline)-PBS, and the results were compared without adding inhibitors. 125 The accumulation of I-HPG was determined as Na (Na+-control) and Ch (Ch-control), respectively. Because xCT is a Na+-independent amino acid transporter, Ch-PBS, which does not contain Na+, was used as the buffer solution in the accumulation experiment to eliminate the contribution of the Na+-dependent amino acid transport system. Specifically, LS180 or DLD-1 is 1.0 x 10 5 Cells were seeded per well, the culture medium in each well was removed, and each inhibitor and buffer solution adjusted to a final concentration of 1.0 mM were added. After 30 seconds, 125 I-HPG was administered at 18.5 kBq / well. Five minutes after administration, the cell surface and wells were washed twice with Na+-PBS or Ch-PBS, and the cells were lysed with 0.1 N NaOH. Radioactivity was measured using an Autowell γ counter. After measurement, the amount of cellular protein in each well was measured using a Multiskan FC absorbance microplate reader (Thermo Scientific). 125 The accumulation amount of I-HPG was corrected. The composition of Na-PBS (Na-phosphate buffer) was 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4·12H2O, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.9 mM CaCl2·2H2O, and 0.5 mM MgCl2. The composition of Ch-PBS (choline-phosphate buffer) was such that Na was replaced with Ch (137 mM choline chloride, 2.7 mM KCl, 8 mM Na2HPO4·12H2O, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.9 mM CaCl2·2H2O, 0.5 mM MgCl2).

[0048] (result) In the loading of specific inhibitors of amino acid transport systems 125 The accumulation rate of I-HPG in LS180 is shown in Figure 6. The accumulation rate of the control in the presence of Na+ ("Na" in Figure 6) is expressed as 100%. The contribution of System A was confirmed to be less than 10% based on the inhibitory effect of MeAIB in the presence of Na ("Na MeAIB" in Figure 6) and in the absence of Na ("Ch MeAIB" in Figure 6). Furthermore, the contribution of System Xc- was confirmed to be 64% with SSZ inhibition and 50% with Cys-Cys inhibition. In the loading of specific inhibitors of amino acid transport systems 125 The accumulation rate of I-HPG in DLD-1 is shown in Figure 7. The control accumulation rate in the presence of Na+ ("Na" in Figure 7) is expressed as 100%. No inhibitory effects were observed with MeAIB ("Ch MeAIB" in Figure 7) and BCH ("Ch BCH" in Figure 7), confirming that only System Xc- contributed to the accumulation rate. Furthermore, the contribution of System Xc- was confirmed to be 64% with SSZ inhibition and 40% with Cys-Cys inhibition. These results suggest that the xCT transport fraction was similarly high in both cell lines, regardless of the amount of xCT gene expression. 125 I-HPG has been shown to be useful.

[0049] (General remarks) The xCT functional activity measuring agent and the xCT functional activity testing method of the present invention measure xCT functional activity, which is not necessarily proportional to the amount of xCT gene expression, in cells (cancer cells) (particularly, per cell or per transporter). 125It can be calculated based on the amount of I-HPG accumulated. For example, xCT gene expression may be reduced but functional activity may be increased, which may contribute to the redox regulation of cancer cell survival. Furthermore, xCT functional activity may be reduced or lost even when xCT gene expression is increased or maintained. Furthermore, xCT functional activity may be altered by irradiation. The present inventors have also succeeded in calculating the contribution of System Xc- using inhibitors AIB and NEM. However, the method of calculating the contribution of System Xc- using inhibitors of System Xc- (particularly sulfasalazine (SSZ) or cystine (Cys-Cys)) of the present invention can detect the contribution of System Xc- with higher sensitivity and accuracy than the method of calculating the contribution of System Xc- using AIB and NEM. [Industrial Applicability]

[0050] As described above, the xCT functional activity measuring agent of the present invention can be used to measure xCT functional activity. Furthermore, it can also measure xCT functional activity after X-ray irradiation, making it possible to measure xCT functional activity that cannot be predicted from the amount of xCT gene expression. This makes it possible to confirm whether cancer is activated. Furthermore, it is useful because it can also determine the therapeutic effect of xCT inhibitors, which is expected to lead to the development of new anticancer drugs. Furthermore, it can also be used in cancer diagnosis.

Claims

1. 125 An agent for measuring xCT functional activity, comprising l-4-hydroxyphenylglycine.

2. A method for testing or assisting in testing xCT functional activity, comprising the steps of: (1) administering the xCT functional activity measuring agent according to claim 1 to a subject or a test sample; (2) detecting the administered xCT functional activity measuring agent; (3) determining the xCT functional activity from the detection results;

3. 125 A cancer radioactive diagnostic agent containing I-4-hydroxyphenylglycine.

4. A cancer testing method or testing assistance method, comprising the following steps: (1') administering the radioactive diagnostic agent according to claim 3 to a subject or a test sample; (2') detecting the administered radioactive diagnostic agent; (3') A step of determining the presence or absence of cancer based on the detection results.

5. 125 A kit for measuring xCT functional activity and / or diagnosing cancer, comprising l-4-hydroxyphenylglycine.

6. For xCT 125 A method for testing xCT functional activity, comprising a step of measuring the rate of accumulation of l-4-hydroxyphenylglycine in cancer cells.

7. The method for testing xCT functional activity according to claim 6, further comprising the steps of: (A) administering the xCT functional activity measuring agent according to claim 1 to a test sample in the absence of Na+; (B) Na + into cancer cells in the absence 125 The accumulation rate of I-4-hydroxyphenylglycine was + In the absence of System Xc-inhibitors, 125 A process of calculating the contribution rate of System Xc-, which is the value obtained by subtracting the accumulation rate of I-HPG.

8. Represented by the following formula (II): 125 I-4-hydroxyphenylglycine. [Chemical Formula I]

9. A method for screening an xCT functional activity inhibitor or an xCT functional activity promoter, comprising the steps of: (1) 125 administering an xCT functional activity measuring agent containing l-4-hydroxyphenylglycine and a test compound to a test sample; (2) detecting the administered xCT functional activity measuring agent; (3) A step of determining, based on the detection results, a test compound that reduces xCT functional activity as an xCT functional activity inhibitor, or a test compound that improves xCT functional activity as an xCT functional activity promoter.

10. An xCT functional activity measuring device comprising the following means: 1) Included in the test sample 125 A method for measuring the accumulation of I-4-hydroxyphenylglycine 2) Measured by the method 125 A method for calculating the contribution rate of System Xc-, an index of xCT functional activity, from the accumulation of l-4-hydroxyphenylglycine

11. A program for measuring xCT functional activity, Computer 125 I- A program that functions as a means of calculating the contribution rate of System Xc-, an indicator of xCT functional activity, from the amount of 4-hydroxyphenylglycine accumulated.

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