Deuterium-labeled fumaric acid or salt thereof for detecting cancer cell death

Abstract

The invention provides a use of deuterium-labeled fumaric acid or a salt thereof for detecting cancer cell death. The invention provides an application of deuterium-labeled fumaric acid or a salt thereof in preparation of a medicine or a kit. The medicine or the kit is used for detecting death of cancer cells in a subject, detecting a tumor position in the subject or screening an anti-cancer medicine.

Description

[0001] This application claims priority to a Chinese invention patent application filed on July 31, 2023, with application number 202310957183.5 and titled “Detection of cancer cell death by deuterium-labeled fumaric acid or its salts”. The priority text is incorporated herein by reference. Technical Field

[0002] The invention relates to the field of medical detection, and in particular to use of deuterium-labeled fumaric acid or a salt thereof in preparing medicines or kits. Background Art

[0003] Currently, the response of solid tumors to treatment is mainly assessed based on changes in tumor size. However, changes in tumor size may take weeks to appear after the start of treatment and, in some cases, may not appear at all. For example, in the case of treatments that inhibit tumor growth but do not cause tumor regression, the effectiveness of treatment cannot be judged by measuring tumor size.

[0004] Metabolic changes can give an early indication of treatment response, for example, by measuring 2-[ 18 F]-fluoro-2-deoxy-D-glucose uptake (FDG-PET) is used to assess glycolytic activity. As with FDG-PET, hyperpolarized [1- 13 C]pyruvate imaging can also be used to monitor drug target binding and subsequent tumor cell death.

[0005] While metabolic imaging can indicate drug targeting and, in some cases, tumor cell death, a more direct method of monitoring tumor cell death is needed to provide an indication of long-term treatment outcomes. Summary of the invention

[0006] In response to the above problems, the inventors detected the content of fumaric acid or its salts and malic acid or its salts by magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging, and verified for the first time that the dynamic content changes of fumaric acid-malic acid reflect the death of cancer cells.

[0007] In one aspect, the present application provides the use of deuterium-labeled fumaric acid or a salt thereof in the preparation of a drug or a kit for detecting the death of cancer cells in a subject, for detecting the location of a tumor in a subject, or for screening anticancer drugs or predicting a subject's response to an anticancer drug or radiotherapy.

[0008] In one embodiment, the medicament or kit is for use in magnetic resonance spectroscopy, magnetic resonance imaging, or deuterium metabolism imaging.

[0009] In one embodiment, deuterium-labeled fumaric acid or a salt thereof is administered to a subject, and the signals of fumaric acid or a salt thereof, malic acid or a salt thereof, and water in the subject's cancer tissue are detected by magnetic resonance spectroscopy, magnetic resonance imaging, or deuterium metabolic imaging. The death of cancer cells is determined, the location of the tumor is detected, or anticancer drugs are screened based on the changes in the content of fumaric acid or a salt thereof and / or malic acid or a salt thereof.

[0010] In one embodiment, magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging is performed 1 min-24 h after administration of deuterium-labeled fumaric acid or a salt thereof, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 1.5 h, 2 h, 2.5 h, 5 h, 10 h, 15 h, 20 h after administration of deuterium-labeled fumaric acid or a salt thereof.

[0011] In one embodiment, the drug or kit further comprises an anti-cancer drug, and the anti-cancer drug is administered before or after the deuterium-labeled fumaric acid or a salt thereof is administered to the subject.

[0012] In one embodiment, the presence of the following indications after administration of an anticancer drug to a subject indicates that cancer cells in the subject are dead, that the administered anticancer drug is effective in killing cancer cells in the subject, or that the number of cancer cells in the subject is reduced, compared to a subject with the same cancer who is not administered the anticancer drug or before administration of the anticancer drug:

[0013] A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0014] The concentration of deuterium-labeled fumaric acid or a salt thereof is reduced by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0015] The proportion of deuterium-labeled malic acid or its salt to the total malic acid or its salt content is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0016] The ratio of the signal intensity of deuterium labeled malic acid or a salt thereof to deuterium labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times; and / or

[0017] 400, 500, 600, 700, 800, 900, 1000-fold.

[0018] In one embodiment, after administration of an anticancer drug, the location where the deuterium-labeled malic acid or salt thereof signal appears in the subject may be the location of a tumor.

[0019] In one embodiment, deuterium-labeled fumaric acid or a salt thereof is administered orally or by injection, such as intramuscular or intravenous injection.

[0020] In one embodiment, the medicament or kit further contains a pharmaceutically acceptable salt or excipient.

[0021] In one embodiment, the dosage of deuterium-labeled fumaric acid or its salt administered is 0.1-100 mg / kg, for example 0.1, 0.25, 0.5, 1, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100 mg / kg based on the subject's body weight; in a preferred embodiment, it is 75 mg / kg.

[0022] In one embodiment, the deuterium-labeled fumaric acid or its salt contains at least two deuterium atoms; in a preferred embodiment, the deuterium-labeled fumaric acid is [2,3,-d2]-fumaric acid. The structural formula of the deuterium-labeled fumaric acid can be:

[0023]

[0024] In one embodiment, the deuterium-labeled fumarate is deuterium-labeled disodium fumarate; in a preferred embodiment, the deuterium-labeled disodium fumarate is [2,3,-d2]-disodium fumarate.

[0025] In one embodiment, deuterium-labeled fumaric acid is prepared by:

[0026] Step 1, synthesis of [2,3,-d2]-dimethyl fumarate: dissolving dimethyl butynoate in an appropriate amount of an organic solvent, adding an appropriate amount of heavy water, adding triphenylphosphine to the reaction system in batches, reacting at an appropriate temperature, and treating after the reaction to obtain [2,3,-d2]-dimethyl fumarate;

[0027] Step 2, synthesis of [2,3,-d2]-fumaric acid: add the [2,3,-d2]-dimethyl fumarate prepared in the previous step to a suitable solvent, add a suitable base, react at a suitable temperature, treat the crude product after the reaction is completed, and then recrystallize to obtain the target compound, i.e., [2,3,-d2]-fumaric acid.

[0028] In one embodiment, the solvent in the above step 1 includes tetrahydrofuran, methanol, acetone, 1,4-dioxane, ethyl acetate, dichloromethane and toluene, and in a preferred embodiment, tetrahydrofuran.

[0029] In one embodiment, the amount of solvent used in step 1 is 1-10 mol / L, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mol / L.

[0030] In one embodiment, the amount of deuterated water in step 1 above includes 0.1-5 eq, for example 0.1, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5 eq; in a preferred embodiment, it is 1.5 eq.

[0031] In one embodiment, the suitable temperature in the above step 1 is 20-100°C; in a preferred embodiment, it is 60-80°C, for example 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80°C.

[0032] In one embodiment, the suitable alkali in the above step 2 may be 10%-12% potassium hydroxide; in a preferred embodiment, it is 10% potassium hydroxide.

[0033] In one embodiment, the suitable temperature in the above step 2 is 20-100°C, and in a preferred embodiment it is 80-100°C; for example 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100°C.

[0034] In one embodiment, the cancer is a solid tumor.

[0035] In one embodiment, the solid tumor is selected from breast cancer, colon cancer, lymphoma, lung cancer, liver cancer, rectal cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma or mesothelioma; in a preferred embodiment, the solid tumor is breast cancer, colon cancer or lymphoma.

[0036] In one embodiment, the subject is a mammal, such as a human.

[0037] In one aspect, the present invention provides a method for screening an anticancer drug, comprising:

[0038] a. administering the drug to be tested to the cancer disease model;

[0039] b. Extracting cancer tissue cells from cancer disease models;

[0040] c. adding deuterium-labeled fumaric acid or a salt thereof to the suspension of extracted cells; and

[0041] d. Detecting signals of fumaric acid or its salt, malic acid or its salt and water in the cell culture by magnetic resonance spectroscopy.

[0042] In one embodiment, the following indications are indicative of the drug being tested being a drug of interest when compared to cells of the same tissue of the same disease model as a control to which the drug has not been administered:

[0043] A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0044] The concentration of deuterium-labeled fumaric acid or a salt thereof is reduced by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0045] The proportion of deuterium-labeled malic acid or its salt to the total malic acid or its salt content is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0046] The ratio of the signal intensity of deuterium labeled malic acid or a salt thereof to deuterium labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times; and / or

[0047] 400, 500, 600, 700, 800, 900, 1000-fold.

[0048] In one embodiment, step a and step b are separated by 1 h-36 h, for example 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h.

[0049] In one embodiment, step c and step d are separated by 1 min-24 h, for example 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 1.5 h, 2 h, 2.5 h, 5 h, 10 h, 15 h, 20 h.

[0050] In one embodiment, the amount of deuterium-labeled fumaric acid or its salt added in step c is 0.1-100mM, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM.

[0051] In one embodiment, the deuterium-labeled fumaric acid or a salt thereof is the same as the deuterium-labeled fumaric acid or a salt thereof in the above aspects.

[0052] In one embodiment, the cancer is the same as in the above aspects.

[0053] In one embodiment, the disease model is a mammal, such as a human.

[0054] In one aspect, the present application provides a method for screening anticancer drugs, comprising:

[0055] a. administering the drug to be tested to the cancer disease model;

[0056] b. administering deuterium-labeled fumaric acid or a salt thereof to a cancer disease model; and

[0057] c. Detecting signals of fumaric acid or its salt, malic acid or its salt, and water in cancer tissue of a cancer disease model by magnetic resonance spectroscopy, magnetic resonance imaging, or deuterium metabolic imaging.

[0058] In one embodiment, the following indications are indicative of a test drug being a drug of interest when compared to the same tissue in the same disease model as a control but without drug administration:

[0059] A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0060] The concentration of deuterium-labeled fumaric acid or a salt thereof is reduced by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0061] The proportion of deuterium-labeled malic acid or its salt to the total malic acid or its salt content is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0062] The ratio of the signal intensity of deuterium labeled malic acid or a salt thereof to deuterium labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times; and / or

[0063] 400, 500, 600, 700, 800, 900, 1000-fold.

[0064] In one embodiment, step a and step b are separated by 1 h-36 h, for example 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h.

[0065] In one embodiment, step b and step c are separated by 1 min-24 h, for example 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 1.5 h, 2 h, 2.5 h, 5 h, 10 h, 15 h, 20 h.

[0066] In one embodiment, the dosage of deuterium-labeled fumaric acid or its salt administered in step b is 0.1-100 mg / kg, such as 0.1, 0.25, 0.5, 1, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100 mg / kg based on the body weight of the cancer disease model; in a preferred embodiment, it is 75 mg / kg.

[0067] In one embodiment, the deuterium-labeled fumaric acid or a salt thereof in step b is administered to the cancer disease model orally or by injection, such as intravenous or intramuscular injection.

[0068] In one embodiment, the deuterium-labeled fumaric acid or a salt thereof is the same as the deuterium-labeled fumaric acid or a salt thereof in the above aspects.

[0069] In one embodiment, the cancer is the same as in the above aspects.

[0070] In one embodiment, the disease model is a mammal, such as a human.

[0071] In one aspect, the present application provides a method for screening anticancer drugs, comprising:

[0072] a. administering the test drug to the cancer disease model, and performing magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging on the disease model before and after the administration of the test drug, wherein deuterium-labeled fumaric acid or a salt thereof is administered to the cancer disease model before the detection; and

[0073] b. Detecting signals of fumaric acid or its salt, malic acid or its salt, and water in cancer tissue of a cancer disease model by magnetic resonance spectroscopy, magnetic resonance imaging, or deuterium metabolic imaging.

[0074] In one embodiment, the following indications are indicative of the test drug being the target drug compared to before administration of the test drug:

[0075] A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0076] The concentration of deuterium-labeled fumaric acid or a salt thereof is reduced by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0077] The proportion of deuterium-labeled malic acid or its salt to the total malic acid or its salt content is increased by at least 0.1-100 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100 times;

[0078] The ratio of the signal intensity of deuterium labeled malic acid or a salt thereof to deuterium labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times, for example, by at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times; and / or

[0079] 400, 500, 600, 700, 800, 900, 1000-fold.

[0080] In one embodiment, the detection in step a is carried out 1 min-24 h after administration of deuterium-labeled fumaric acid or a salt thereof, for example 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 1.5 h, 2 h, 2.5 h, 5 h, 10 h, 15 h, 20 h later.

[0081] In one embodiment, in step a, the disease model is tested 1 h-36 h before and / or 1 h-36 h after administration of the test drug, for example, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h before or after administration.

[0082] In one embodiment, the dosage of deuterium-labeled fumaric acid or its salt administered in step a is 0.1-100 mg / kg, such as 0.1, 0.25, 0.5, 1, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100 mg / kg based on the body weight of the cancer disease model; in a preferred embodiment, it is 75 mg / kg.

[0083] In one embodiment, the deuterium-labeled fumaric acid or a salt thereof in step a is administered to the cancer disease model orally or by injection, such as intravenous or intramuscular injection.

[0084] In one embodiment, the deuterium-labeled fumaric acid or a salt thereof is the same as the deuterium-labeled fumaric acid or a salt thereof in the first aspect.

[0085] In one embodiment, the cancer is the same as the cancer in the first aspect.

[0086] In one embodiment, the disease model is a mammal, such as a human.

[0087] In one aspect, an in vitro method for detecting the response of cells to radiation therapy is provided, which comprises irradiating cells, then adding deuterium-labeled fumaric acid to the cells, measuring the concentration of fumaric acid, malic acid and / or HDO, and within 20 hours, preferably 2 hours after radiation treatment, a decrease in fumaric acid concentration compared to cells not subjected to radiation treatment indicates that radiation therapy effectively kills cells; or,

[0088] Within 2 hours after irradiation, elevated malate concentrations compared to non-irradiated cells indicated effective cell killing over the range of radiation therapy doses.

[0089] In one embodiment, the cell is a glioma cell, such as a U251 cell or a U87 cell.

[0090] In one embodiment, radiation therapy is performed with 5-100 Gly, preferably 5-20 Gly.

[0091] In one embodiment, by 2 The concentrations of fumaric acid, malic acid and / or HDO were determined by H NMR.

[0092] In one embodiment, the deuterium-labeled fumaric acid has the structural formula:

[0093] In all the above aspects, multiple measurements can be performed at multiple time points after the administration or addition of deuterium-labeled fumaric acid or a salt thereof, for example, once every 0.5min-1h, for example, every 0.5min, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 1h.

[0094] Advantages of the present invention include:

[0095] 1. The present invention provides a sensitive method for non-invasive detection of cancer cell death in vivo, which has important clinical significance for patient condition monitoring, treatment response evaluation and subsequent medication.

[0096] 2. The method provided by the present invention can achieve drug screening, patient screening, tumor location detection, tumor peripheral lymph node determination, tumor volume prediction, etc. based on the detection of cancer cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 This is a flow chart of one embodiment of the present application, wherein drugs are screened by in vitro testing.

[0098] Figure 2 The present invention is a flowchart of an embodiment of drug screening for the present invention, wherein cancer cell death is monitored by in vivo DMI scanning to screen anticancer drugs.

[0099] Figure 3 The present invention is a flowchart of an embodiment of drug screening of the present invention, wherein the effectiveness of the anticancer drug is determined by detecting the death of cancer cells before and after administration of the anticancer drug, thereby screening the anticancer drug.

[0100] Figure 4 The technical route of the cell experiment of the embodiment is shown.

[0101] Figure 5 A graph showing the changes in fumaric acid concentration over time in U87 cells and U251 cells is shown.

[0102] Figure 6 A graph showing the change in malate concentration over time for U87 cells and U251 cells.

[0103] Figure 7 A graph showing the change in HDO concentration over time for U87 cells and U251 cells is shown. DETAILED DESCRIPTION

[0104] In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Although it is believed that those of ordinary skill in the art fully understand the following terms, the following definitions are still stated to help illustrate the subject matter disclosed by the present invention.

[0105] In one embodiment of the present invention, the detection of deuterium-labeled fumaric acid or its salt in the cell suspension is performed by magnetic resonance spectroscopy. The cells in the suspension treated with the drug to be tested are placed on a plate, treated with the anticancer drug for 24 h, washed with PBS, and then incubated in 10 mL of culture medium at 1×10 6 Resuspend cells / mL and add 5mM deuterium-labeled fumaric acid or its salt. Take 1ml sample at the specified time point and obtain 2 H NMR. The resonance amplitudes of water, fumaric acid or its salts and malic acid or its salts were normalized to the DMSO-d6 peak, correcting for the slight saturation of the DMSO-d6 resonance, to calculate the concentrations. The absolute concentrations were obtained by correcting for the number of deuterons per molecule.

[0106] In one embodiment of the present invention, the detection of deuterium-labeled fumaric acid or its salt in vivo is achieved by magnetic resonance imaging, and the experiment is performed at 7T, using a 72mm diameter birdcage volume coil for H transmission and reception, and a 10mm diameter single-loop surface coil located above the tumor for 2 H transmission and reception. Axial 'H' images acquired with a fast spin echo (FSE) pulse sequence to localize the tumor. Continuous 2 H spectra were acquired using a pulsed acquisition sequence with 2 ms BIR4 adiabatic excitation pulses, a nominal flip angle of 67°, a repetition time (TR) of 140 ms, and a spoiler gradient in the z-axis to eliminate any residual transverse magnetization. Thirteen 5-min spectra were acquired over 65 min. Localization of the signal to the tumor was confirmed by excitation curves and chemical shift images from the surface coil, indicating that the signal was primarily confined to the tumor. After peak fitting, the HDO peak intensity at the first time point was assumed to correspond to the peak in the HDO. 2 The natural abundance of H in tissues is estimated 2The abundance of H is 10.12 mM. The HDO signal provides an internal reference for estimating the concentration of deuterium-labeled fumaric acid or its salts. By assuming that the in vivo T 1 s and T measured in vitro at 14.1T 1 s similar to that of deuterium-labeled fumaric acid or its salts 2 The number of H nuclei was corrected for saturation (15%). It was assumed that malic acid or its salts had a similar T value to fumaric acid or its salts. 1 , the concentration of malic acid or its salt is estimated from the intensity of the upfield resonance. In vivo, the downfield resonance overlaps with the HDO peak, resulting in a slight overestimation of the labeled water concentration at later time points. Three-dimensional chemical shift imaging (CSI) was described in F. Kreis et al., Measuring tumor glycolytic flux in vivo by using fast deuterium MRI. Radiology 294, 289–296 (2020).

[0107] In one embodiment of the present invention, the magnetic resonance results are statistically and graphically analyzed using Prism v8 (GraphPad). Unless otherwise stated, the results for multiple time points are expressed as mean ± standard deviation.

[0108] As described herein, the term "anticancer drug" refers to drugs that resist cancer, including but not limited to anti-microtubule drugs, such as paclitaxel, docetaxel, albumin paclitaxel; anti-metabolism drugs, such as gemcitabine, vinorelbine; folate reductase inhibitors, such as pemetrexed; targeted drugs, i.e., targeted at changes in genes in the body, including driver genes EGFR, ALK, ROS1, and HER-2 genes of breast cancer, etc., targeted at changes in these genes are called targeted drugs, including gefitinib, alectinib, etc.; drugs that inhibit angiogenesis in the body, such as bevacizumab, apatinib, anlotinib; and immune checkpoint inhibitors, such as inhibitors for PD-1, PD-L1, etc. The method, use or reagent of the present invention can be used to evaluate the anticancer effect of a subject after administration of an anticancer drug.

[0109] The anticancer drug may be a chemotherapeutic agent, examples of which include erlotinib (Genentech / OSI Pharm.), bortezomib (Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (AstraZeneca), sunitib (Pfizer / Sugen), letrozole (Novartis), imatinib mesylate (Novartis), finasunate (Novartis), oxaliplatin (Sanofi), 5-FU (5-fluorouracil), folinic acid, rapamycin (sirolimus, Wyeth), lapatinib (GSK572016, Glaxo Smith Kline), lonafamib (SCH66336), sorafenib (Bayer Labs), gefitinib (AstraZeneca), AG1478, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine. lomelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors (including finasteride and dutasteride); vorinostat, romidepsin, panbicin, valproic acid, mocetinostat dolastatin; aldesleukin, talcum duocarcin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin;sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, clofosamide, estramustine, ifosfamide, mechlorethamine, methoxychloramine hydrochloride, melphalan, novembichin, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzoxazone, fermustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl. 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and the new carcinogens and related chromophore proteins (enediyne antibiotic chromophores), aclacinomycins, actinomycins, authramycins, azaserines, bleomycins, cactinomycins, carabicins, caminomycins, carzinophilins, chromomycins, dactinomycins, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidino-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), bicin), esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olenomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as caprotestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2",2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine);urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chlorambucil ( chloranmbucil); (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinols, such as retinoic acid; and any pharmaceutically acceptable salts, acids and derivatives thereof. Chemotherapeutic agents also include "platinum" chemotherapeutic agents, which contain organic compounds containing platinum as a component of the molecule. Typically, platinum chemotherapeutic agents are coordination complexes of platinum. Platinum chemotherapeutic agents are sometimes referred to as "platinum drugs" in the art. Examples of platinum chemotherapeutic agents include, but are not limited to, carboplatin, cisplatin and oxaliplatin. ;

[0110] Chemotherapeutic agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (Genentech); cetuximab (Imclone); panitumumab (Amgen), rituximab (Genentech / Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (Wyeth). Other humanized monoclonal antibodies with therapeutic potential for combination with the compounds of the present invention include: apolizumab, aselizumab, alizumab, barbiturumab, bivatuzumabmertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin), ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab , pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sirolimusumab, cilizumab, sontuzumab, tacatuzumabChemotherapeutic agents include EGFR inhibitors and tyrosine kinase inhibitors.

[0111] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "Comprising" is a technical term used in claim language, meaning that the elements are present, but other elements may also be added and still form a structure or method within the scope of the claim.

[0112] As used herein, the terms "a," "an," and "the" mean "one or more" when used in this application, including the claims.

[0113] Unless otherwise indicated, all numbers expressing quantities of size, reaction conditions, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the presently disclosed subject matter.

[0114] As used herein, when referring to a value or amount of size (i.e., diameter), weight, concentration, or percentage, the term "about" is intended to include variations of ±20% or ±10% in one embodiment, ±5% in another embodiment, ±1% in another embodiment, and ±0.1% in yet another embodiment relative to the stated amount, such that these variations are suitable for performing the disclosed methods.

[0115] As used herein, when used in the context of a list of entities, the term "and / or" refers to the entities present individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, and also includes any and all combinations and subcombinations of A, B, C, and D.

[0116] As used herein, the phrase "consisting of does not include any elements, steps, or ingredients not specified in a claim.

[0117] As used herein, the term "subject" refers to a mammal, particularly a human patient, for example, a human patient suffering from cancer. The terms "subject" and "patient" are used interchangeably herein.

[0118] The use of deuterium-labeled fumaric acid or its salt herein can be achieved by any suitable method and technology currently or expectedly known to those skilled in the art. For example, deuterium-labeled fumaric acid or its salt can be formulated in a physiologically or pharmaceutically acceptable form and used by any suitable route known in the art, including, for example, oral and injection routes. As used herein, the term injection includes, for example, subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal administration by injection. The use of the disclosed deuterium-labeled fumaric acid or its salt can be a single administration, or can be used at continuous or different intervals as those skilled in the art can easily determine.

[0119] Deuterium-labeled fumaric acid or its salt disclosed herein can be formulated to be combined with suitable carriers and / or excipients, thereby facilitating its effective administration. The carrier used and / or can also be in various forms. These include, for example, solid, semisolid and liquid forms. The preferred form depends on the expected mode of administration and application.

[0120] Deuterium-labeled fumaric acid or its salt can be present in unit dose or multi-dose containers, such as sealed ampoules and vials. It should be understood that, in addition to the carriers and / or excipients specifically mentioned above, the medicament or kit disclosed herein may also include other reagents conventional in the art related to the type of preparation under consideration.

[0121] For oral administration, deuterium-labeled fumaric acid or a salt thereof may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, suspensions, syrups, wafers, aerosol sprays, and the like.

[0122] Tablets, lozenges, pills, capsules, etc. may also contain the following substances: binders, such as tragacanth, acacia, corn starch or gelatin; diluents, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose or aspartame, or flavorings, such as peppermint, wintergreen oil or cherry flavorings may be added. When the unit dosage form is a capsule, in addition to the above types of materials, it may also contain a liquid carrier, such as vegetable oil or polyethylene glycol. Various other materials may exist as a coating or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac, or sugar, etc. Syrups may contain active compounds, sucrose or fructose (as sweeteners), methylparabens and propylparabens (as preservatives), dyes and flavorings, such as cherry or orange flavorings. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.

[0123] Deuterium-labeled fumaric acid or its salt disclosed herein can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of deuterium-labeled fumaric acid or its salt can be prepared in water optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and their mixtures and oils. Under conventional storage and use conditions, these preparations can include a preservative for preventing microbial growth.

[0124] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder containing the active ingredient, which is suitable for temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be a sterile fluid and is very stable under manufacturing and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and their suitable mixtures. Suitable fluidity can be, for example, by forming liposomes, by maintaining the desired particle size in the case of dispersions, or by using a surfactant to maintain. Optionally, the prevention of microbial action can be achieved by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents are preferably included, such as sugars, buffers, or sodium chloride. The long-acting absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0125] Sterile injectable solutions are prepared as follows: deuterium-labeled fumaric acid or its salt disclosed herein is introduced into an appropriate solvent in the required amount together with various other above-listed ingredients as required, and then filtered sterilized. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient and any additional desired ingredients present in the previously sterile-filtered solution.

[0126] As used herein, the term "buffer" refers to a pharmaceutically acceptable buffer. The term "buffer" encompasses those agents that maintain the pH of a solution within an acceptable range, for example, and includes, but is not limited to, acetate, histidine, (tris(hydroxymethyl)aminomethane), citrate, succinate, glycolate, etc. Generally, a "buffer" as used herein has a pKa and a buffering capacity suitable for a pH range of about 5 to about 6, preferably about 5.5.

[0127] As used herein, "surfactants" are compounds that are commonly used in pharmaceutical formulations to prevent drugs from being adsorbed to surfaces and / or aggregating. In addition, surfactants reduce the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, exemplary surfactants can significantly reduce surface tension when present at very low concentrations (e.g., 5% w / v or less, such as 3% w / v or less, such as 1% w / v or less, or such as 0.4% w / v or less, such as less than 0.1% w / v or less, such as 0.04% w / v). Surfactants are amphiphilic, meaning that they are typically composed of hydrophilic and hydrophobic or lipophilic groups and are therefore able to form micelles or similar self-assembled structures in aqueous solution.Known pharmaceutical surfactants include glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin (anionic surfactants); benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids (cationic surfactants); and alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates. Stearate, polyethylene glycol esters (polyoxyl hydroxystearate), polyethylene glycol glycerides (polyoxylglycerides), polysorbates (polysorbate 20 or polysorbate 80), propylene glycol dilaurate (propylene glycol dilaurate), propylene glycol monolaurate (propylene glycol monolaurate), sorbitan esters sucrose stearate (sucrose stearate), tricaprylin (tricaprylin) and TPGS (nonionic and zwitterionic surfactants).

[0128] As used herein, "diluent" is a diluent that is pharmaceutically acceptable (safe and non-toxic to human administration) and can be used to prepare a pharmaceutical composition or pharmaceutical formulation (the terms "composition" and "formulation" are used interchangeably herein). Preferably, such diluents of the composition only dilute the antibody concentration without diluting the buffer and stabilizer. Therefore, in one embodiment, the diluent contains the same concentration of buffer and stabilizer as present in the pharmaceutical composition of the present invention. Another exemplary diluent includes sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution preferably of an acetate buffer, a sterile physiological saline solution, Ringer's solution, or a dextrose solution. In one embodiment, the diluent comprises an acetate buffer and sorbitol or consists essentially of an acetate buffer and sorbitol.

[0129] As used herein, the term "preservative" refers to an antimicrobial substance that inhibits the activity of microorganisms, including but not limited to α-terpineol, methylisothiazolinone, chloroxylenol, hexachlorophene, chlorhexidine and other cationic biguanides, methylene chloride, iodine and iodophores, triclosan, 2-aminoethylsulfonamide (taurinamide), nitrofurantoin, methenamine, aldehydes, acrylic acid, silver, benzyl peroxide, alcohols, and carboxylic acids and salts. It will be appreciated by those skilled in the art that these preservatives can be used in combinations of two or more to obtain a synergistic or additive effect. Some examples of combinations of preservatives include chlorhexidine, chlorhexidine and chloroxylenol, chlorhexidine and methylisothiazolinone, chlorhexidine and (α-terpineol, a mixture of methylisothiazolinone and α-terpineol; thymol and chloroxylenol; chlorhexidine and cetylpyridinium chloride; or chlorhexidine, methylisothiazolinone and thymol. These combinations provide a broad spectrum of activity against a variety of organisms.

[0130] The disclosed medicine and / or kit may optionally include a pharmaceutically acceptable carrier and / or excipient. In one embodiment, the medicine and / or kit include one or more other components, adjuvants or adjuvants as described herein. In another embodiment, the medicine and / or kit include one or more other active ingredients, such as those described herein. In one embodiment, the medicine and / or kit include instructions or packaging materials for explaining how to apply deuterium-labeled fumaric acid or its salt or how to perform DMI. The container of the medicine and / or kit can be any suitable material, such as glass, plastic, metal, etc., and is any suitable size, shape or structure. In one embodiment, deuterium-labeled fumaric acid or its salt disclosed herein is provided as a solid in the medicine and / or kit, such as a tablet, pill or powder form. In another embodiment, deuterium-labeled fumaric acid or its salt disclosed herein is provided as a liquid or solution in the kit.

[0131] Chemical shift imaging (CSI) is described in de Feyter, et al., Science advances, 20184(8):eaat7314, which is incorporated herein by reference. Echo planar spectroscopy imaging (EPSI) can also be used (Mansfield P. Magnetic Resonance in Medicine. 1984 1(3):370-86, which is incorporated herein by reference). There are also newer implementations of EPSI (Chen AP, et al. Magn Reson Med. 200758(6):1099-106; Jakob PM, et al. Magnetic resonance in medicine).

[0132] 1995 33(4):573-8, which is incorporated herein by reference).

[0133] An alternative method for producing chemically selective images is to use cumulative phase and multiple sampling windows to produce images that can be combined to produce chemically selective images, which is generally referred to as the Dixon method (Dixon Wt. Radiology. 1984 153 (1): 189-94, which is incorporated herein by reference). This technique uses two images to produce in-phase and out-of-phase combinations of two metabolites. It can also be applied to 3 resonances by using more echoes (Szumowski J, et al. Radiology. 1994 192 (2): 555-61, which is incorporated herein by reference to teach this method). More complex methods can use even more echoes (Wang Jx, et al. Magnetic Resonance in Chemistry. 2016 54 (8): 665-73, which is incorporated herein by reference).

[0134] An alternative approach uses selective excitation of the desired resonance followed by imaging to produce a chemically selective image. Long selective pulses can be used to excite a single resonance peak (Meyer CH, et al., Magnetic Resonance in Medicine. 1990 15(2):287-304, incorporated herein by reference), but these pulses suffer from RF uniformity issues.

[0135] Another suitable method for imaging combines selective excitation with steady-state free precession (SSFP) to rapidly acquire images (Scheffler K, et al. Eur Radiol. 2003 13(11):2409-18, which is incorporated herein by reference). These image sequences are rapid and produce excellent in-plane resolution.

[0136] DMI is hindered by the short T2 of the nucleus. Methods using very short echoes can also be applied in some cases (Hafner S. Magn Reson Imaging. 1994 12 (7): 1047-51; Robson MD et al., Journal of Computer Assisted Tomography. 2003 27 (6): 825-46, which are incorporated herein by reference). A similar technique is scanning imaging (Idiyatullin D, et al. Journal of Magnetic Resonance. 2006 181 (2): 342-9, which is incorporated herein by reference).

[0137] Each of these approaches focuses on producing images that separate the HDO from other resonances, i.e., any other resonances that could contaminate the image, reducing its diagnostic power.

[0138] In some embodiments, the method involves a single imaging step that detects elevated HDO in a tissue, for example by comparing it to other tissues. In other embodiments, the method includes imaging the subject at multiple time points to determine the rate of HDO enrichment in the subject's tissue to detect regional differences in glycolytic flux associated with lesions. The use of kinetic modeling to estimate metabolic turnover has been used to study in vivo metabolism in many contexts (Cerdan S, et al. J Biol Chem. 1990 265 (22): 12916-26; Patel AB, et al. Proc Natl Acad Sci USA 2005

[0139] 102(15):5588-93; Sibson NR, et al. Proc Natl Acad Sci USA 1997;94(6):2699-704, which is incorporated herein by reference). These methods measure enrichment as a function of time to quantitatively measure rate. They are more accurate than taking a single image, but require the patient to be scanned for longer periods of time. Minimizing patient scan time is essential because scans longer than 30 minutes are poorly tolerated by patients.

[0140] Several embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

[0141] Example

[0142] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conditions recommended by the normal conditions or manufacturers are carried out. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. Unless otherwise specified, all amounts listed are described in parts by weight based on the gross weight. The application should not be construed as being limited to the specific embodiments described.

[0143] Example 1. Preparation of compounds

[0144] Synthesis of [2,3,-d2]-fumaric acid

[0145] [2,3,-d2]-fumaric acid was prepared as follows:

[0146]

[0147] Step 1, synthesis of [2,3,-d2]-dimethyl fumarate: dissolve dimethyl butynoate in 1 mol / L tetrahydrofuran, add 1.5 eq of heavy water, add triphenylphosphine to the reaction system in batches, keep the reaction at 60-80°C, and after the reaction is completed, treat to obtain [2,3,-d2]-dimethyl fumarate;

[0148] Step 2, synthesis of [2,3,-d2]-fumaric acid: add the [2,3,-d2]-dimethyl fumarate prepared in the previous step to a suitable solvent, add 10% potassium hydroxide, react at 80-100°C, treat the crude product after the reaction is completed, and then recrystallize to obtain the target compound, i.e., [2,3,-d2]-fumaric acid.

[0149] Synthesis of [2,3,-d2]-Disodium Fumarate

[0150] Dissolve 3 g of [2,3-d2]fumaric acid in 8 M NaOH and then freeze-dry to obtain [2,3,-d2]-disodium fumarate.

[0151] Example 2: Construction of animal model

[0152] Tumor transplantation: Mice were subcutaneously injected with EL4, MDA-MB-231, and col205 cells at a dose of 5x10 5 , 7x10 6 and 10x10 6EL4 tumors were cultured for 10 days, and Col205 and MDA-MB-231 tumors were cultured for 14 and 35 days, respectively, before imaging. Animals bearing EL4 tumors were treated with etoposide (67 mg / kg body weight, intraperitoneal injection) or solvent vehicle (normal saline) 48 hours before imaging. Animals bearing MDA-MB-231 or Colo205 tumors were treated with MEDI3039 (0.8 mg / kg body weight, intravenous injection) or solvent vehicle (normal saline) 24 hours before imaging.

[0153] Cell culture: Mouse lymphoma EL4 and human colorectal cancer Colo205 cells were cultured in RPMI medium (Life Technologies) supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Gibco / Thermo Fisher Scientific), and human triple-negative breast cancer MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum.

[0154] Example 3: [2,3,-d2]-fumarate cell experiment

[0155] The technical route of Example 3 is shown in Figure 4 The specific experiment was conducted at Peking University Shenzhen Hospital.

[0156] 1. 8 6-well plates were plated, 4 of which were U251 cells (human glioma cells) and 4 were U87 cells (human brain glioma cells). The bottom area of ​​each well plate was 2 square centimeters. 8000-10000 cells were inoculated in each well. The culture method was adherent culture. The culture medium was selected to be suitable for the growth of human glioma cells, such as DMEM (containing high glucose) medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomyci.

[0157] 2. Cell recovery

[0158] Take out the cell cryopreservation tube from the liquid nitrogen tank or -80℃ freezer and quickly put it into a 37℃ water bath to thaw. Shake gently during the process to completely melt the cryopreservation solution within 1-2 minutes.

[0159] The cell suspension was transferred to a centrifuge tube containing 4 mL of culture medium and centrifuged at 1000 rpm for 5 minutes.

[0160] Discard the supernatant, resuspend the cells in 1 mL of culture medium, inoculate into a culture dish or culture flask containing an appropriate amount of culture medium, and shake gently to evenly distribute the cells.

[0161] The inoculated cells are placed in an incubator for culture. The culture time varies depending on the cell status and usually requires overnight observation of cell adhesion.

[0162] 3. Cell Passaging

[0163] When the cell density reaches 80%-90%, subculture can be performed.

[0164] The old culture medium was discarded, the cells were washed 1-2 times with PBS, and 0.25% trypsin-0.53 mM EDTA solution was added to digest the cells for 1-3 minutes.

[0165] When cells begin to fall off the bottom of the culture dish, add complete medium to terminate digestion and gently pipette to completely remove the cells. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0166] Discard the supernatant, resuspend the cells with an appropriate amount of fresh culture medium, inoculate the cell suspension into a new culture dish or culture flask at a ratio of 1:2-1:3, and add complete culture medium to an appropriate volume.

[0167] The inoculated cells are placed in an incubator and cultured for 2-3 days.

[0168] 4. After the cell culture is completed, the cells are subjected to radiotherapy, and 4 groups are set up for each cell type: control group, 5Gy group, 10Gy group and 15Gy group.

[0169] Deuterium-labeled fumaric acid incubation detection experiment was performed 72 hours after radiotherapy.

[0170] Deuterium-labeled fumaric acid incubation detection experiment specific operation:

[0171] 1. Digest cells in one well of a 6-well plate to measure cell survival after radiotherapy.

[0172] 2. Add 5mM deuterium-labeled fumaric acid [2,3,-d2]-fumaric acid to the remaining 5 wells, and set them as 1min, 30min, 60min, 120min, and 24h sampling groups. The cells are U251 and U87. Drug: deuterium-labeled fumaric acid.

[0173] 3. Determine the concentration of fumaric acid, malic acid, and HDO by 2H-NMR. Detection method: Find the characteristic peaks of fumaric acid, malic acid, and HDO in the NMR spectrum. The NMR signal of the D (deuterium) atom is different from the signal of the H (hydrogen) atom in frequency. Then use the peak area or peak height in the NMR spectrum to quantify the concentration of HDO.

[0174] As shown in Table 1, it shows the cell survival rate.

[0175] Table 1 Cell survival rate after radiotherapy (cell survival rate calculation formula = (number of surviving cells after radiotherapy / total number of cells before radiotherapy) × 100%)

[0176] 0Gy 5Gy 10Gy 15Gy U251 90.5％ 86.5％ 83.3％ 67.7％ U87 90.4％ 83.1％ 80.4％ 68.5％

[0177] Figure 5 The graphs of fumaric acid concentration changes over time in U87 cells and U251 cells are shown. For fumaric acid concentration, at the same radiation dose, the fumaric acid concentration did not change significantly at the five time points of 2h, and decreased at 24h; the fumaric acid consumption was low at 0 and 5Gy, and high at 10 and 15Gy. The graphs of fumaric acid concentration changes in U251 cells and U87 cells are similar.

[0178] Figure 6 The graphs showing the changes in malic acid concentration over time for U87 cells and U251 cells show that malic acid concentration showed an overall positive correlation trend with radiotherapy dose within the 2h time point.

[0179] Figure 7 A graph showing the change in HDO concentration over time for U87 cells and U251 cells is shown. For HDO concentration, both increased over time.

[0180] The 1-deuterium-fumarate cell experiment was also performed as in Example 3 above.

[0181] As shown in Table 2, it shows the cell survival rate.

[0182] Table 2 Cell survival rate after radiotherapy (cell survival rate calculation formula = (number of surviving cells after radiotherapy / total number of cells before radiotherapy) × 100%)

[0183] 0Gy 5Gy 10Gy 15Gy U251 91.5％ 87.5％ 72.3％ 58.7％ U87 92.4 84.1 68.4％ 54.5％

[0184] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of deuterium-labeled fumaric acid or a salt thereof in the preparation of a drug or a kit for detecting cancer cell death in a subject, for detecting the location of a tumor in a subject, for screening anticancer drugs or determining the effective dose of radiotherapy, or for predicting the response of a subject to an anticancer drug or radiotherapy; Preferably, the drug or kit is used for magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolism imaging; Preferably, the deuterium-labeled fumaric acid or its salt is administered to a subject, and the signals of fumaric acid or its salt, malic acid or its salt and water in the cancer tissue of the subject are detected by magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging, and the death of cancer cells, the location of tumors or the screening of anticancer drugs are determined based on the changes in the content of fumaric acid or its salt and / or malic acid or its salt. Preferably, magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging is performed 1 min to 24 h after administration of the deuterium-labeled fumaric acid or a salt thereof, Preferably, wherein the deuterium-labeled fumaric acid or a salt thereof is administered orally or by injection, such as intramuscular injection or intravenous injection, Preferably, the drug or kit further contains a pharmaceutically acceptable salt or excipient; Preferably, the drug or kit further comprises an anticancer drug, and the anticancer drug is administered before or after the deuterium-labeled fumaric acid or a salt thereof is administered to the subject; Preferably, the following indications appearing after administration of the anticancer drug to the subject indicate that cancer cells in the subject die, the administered anticancer drug is effective in killing cancer cells in the subject, or the number of cancer cells in the subject decreases, compared to a subject not administered the anticancer drug or before administration of the anticancer drug: A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof increases by at least 0.1-100 times; The concentration of deuterium-labeled fumaric acid or its salt is reduced by at least 0.1-100 times; The ratio of the content of deuterium-labeled malic acid or its salt to the total content of malic acid or its salt increases by at least 0.1-100 times; The ratio of the signal intensity of deuterium-labeled malic acid or a salt thereof to that of deuterium-labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times; and / or The ratio of the signal intensity of deuterium-labeled fumaric acid or a salt thereof to that of HDO increases by at least 0.1-1000 times.

2. The use according to claim 1, wherein the administration dose of the deuterium-labeled fumaric acid or its salt is 0.1-100 mg / kg, preferably 75 mg / kg, based on the body weight of the subject.

3. A method for screening anticancer drugs, comprising: a. administering the drug to be tested to the cancer disease model; b. extracting cancer tissue cells from the cancer disease model; c. adding deuterium-labeled fumaric acid or a salt thereof to the extracted cell suspension; and d. detecting signals of fumaric acid or its salt, malic acid or its salt and water in the culture of the cells by magnetic resonance spectroscopy, Preferably, compared with cells of the same tissue of the same disease model to which no drug is administered as a control, the following indications are indicative of the drug to be tested being a target drug: A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof increases by at least 0.1-100 times; The concentration of deuterium-labeled fumaric acid or its salt is reduced by at least 0.1-100 times; The ratio of the content of deuterium-labeled malic acid or its salt to the total content of malic acid or its salt increases by at least 0.1-100 times; The ratio of the signal intensity of deuterium-labeled malic acid or a salt thereof to that of deuterium-labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times; and / or The ratio of the signal intensity of deuterium-labeled fumaric acid or a salt thereof to that of HDO increases by at least 0.1-1000 times; Preferably, wherein: The interval between step a and step b is 1h-36h, The interval between step c and step d is 1 min-24 h, and / or, The amount of deuterium-labeled fumaric acid or its salt added in step c is 0.1-100 mM.

4. A method for screening anticancer drugs, comprising: a. administering the drug to be tested to the cancer disease model; b. administering deuterium-labeled fumaric acid or a salt thereof to the cancer disease model; and c. detecting signals of fumaric acid or its salt, malic acid or its salt and water in the cancer tissue of the cancer disease model by magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging, Preferably, the following indications are indicative of the drug to be tested being a drug of interest, compared to the same tissue of the same disease model as a control to which the drug is not administered: A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof increases by at least 0.1-100 times; The concentration of deuterium-labeled fumaric acid or its salt is reduced by at least 0.1-100 times; The ratio of the content of deuterium-labeled malic acid or its salt to the total content of malic acid or its salt increases by at least 0.1-100 times; The ratio of the signal intensity of deuterium-labeled malic acid or a salt thereof to that of deuterium-labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times; and / or The ratio of the signal intensity of deuterium-labeled fumaric acid or a salt thereof to that of HDO increases by at least 0.1-1000 times.

5. The method according to claim 4, wherein: The interval between step a and step b is 1h-36h, The interval between step b and step c is 1min-24h, Based on the body weight of the cancer disease model, the dosage of the deuterium-labeled fumaric acid or its salt in step b is 0.1-100 mg / kg, preferably 75 mg / kg, and / or, In step b, the deuterium-labeled fumaric acid or its salt is administered to the cancer disease model orally or by injection, such as intravenous or intramuscular injection.

6. A method for screening anticancer drugs, comprising: a. administering a test drug to a cancer disease model, and performing magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging on the disease model before and after administration of the test drug, wherein deuterium-labeled fumaric acid or a salt thereof is administered to the cancer disease model before the detection; and b. detecting the signals of fumaric acid or its salt, malic acid or its salt and water in the cancer tissue of the cancer disease model by magnetic resonance spectroscopy, magnetic resonance imaging or deuterium metabolic imaging, Preferably, compared to before administration of the test drug, the following indications are indicative of the test drug being a target drug: A signal of deuterium-labeled malic acid or a salt thereof is detected, or the concentration of deuterium-labeled malic acid or a salt thereof increases by at least 0.1-100 times; The concentration of deuterium-labeled fumaric acid or its salt is reduced by at least 0.1-100 times; The ratio of the content of deuterium-labeled malic acid or its salt to the total content of malic acid or its salt increases by at least 0.1-100 times; The ratio of the signal intensity of deuterium-labeled malic acid or a salt thereof to that of deuterium-labeled fumaric acid or a salt thereof is increased by at least 0.1-1000 times; and / or The ratio of the signal intensity of deuterium-labeled fumaric acid or a salt thereof to that of HDO increases by at least 0.1-1000 times.

7. The method according to claim 6, wherein: In one embodiment, the detection in step a is performed 1 min to 24 h after administration of deuterium-labeled fumaric acid or a salt thereof, In step a, the disease model is tested 1h-36h before and / or 1h-36h after the administration of the drug to be tested, Based on the body weight of the cancer disease model, the dosage of the deuterium-labeled fumaric acid or its salt in step a is 0.1-100 mg / kg, preferably 75 mg / kg, and / or The deuterium-labeled fumaric acid or its salt in step a is administered to the cancer disease model orally or by injection, for example, by intravenous or intramuscular injection.

8. The use or method according to any one of the preceding claims, wherein the deuterium-labeled fumaric acid or a salt thereof contains at least two deuterium atoms, and / or wherein the deuterium-labeled fumarate is deuterium-labeled disodium fumarate, Preferably, the deuterium-labeled fumaric acid is [2,3,-d2]-fumaric acid. Preferably, the deuterium-labeled fumaric acid has the structural formula: or Preferably, the deuterium-labeled disodium fumarate is [2,3,-d2]-disodium fumarate.

9. The use or method of any of the preceding claims, wherein the cancer is a solid tumor, preferably, the solid tumor is selected from breast cancer, colon cancer, lymphoma, lung cancer, liver cancer, rectal cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, cutaneous fibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma or mesothelioma, preferably breast cancer, colon cancer or lymphoma; Preferably, the subject or disease model is a mammal, preferably a human.

10. An in vitro method for detecting the response of cells to radiotherapy or the effective killing dose, which comprises irradiating cells, then adding deuterium-labeled fumaric acid to the cells, measuring the concentration of fumaric acid and / or malic acid, and within 20 hours, preferably 2 hours after the radiation treatment, a decrease in the fumaric acid concentration compared to cells not subjected to radiation treatment indicates that the radiation therapy effectively kills the cells; and / or, Within 2 hours after irradiation, elevated malate concentrations compared with nonirradiated cells indicate effective cell killing across the radiation therapy dose range; Preferably, the cell is a glioma cell, such as a U251 cell or a U87 cell; Preferably, radiotherapy is performed at 5-100 Gly, preferably 5-20 Gly; Preferably, by 2 The concentration of fumaric acid, malic acid and / or HDO is determined by H nuclear magnetic resonance method; preferably, the deuterium-labeled fumaric acid has the structural formula:

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