AKR1C3 detection method, and diagnostic kit for AKR1C3 detection and its use

Abstract

A method for detecting AKR1C3, a diagnostic kit for detecting AKR1C3, and its use are provided. The method for detecting AKR1C3 includes sequentially treating a formalin-fixed, paraffin-embedded human tissue specimen with an organic solvent, alcohol, and water; antigen retrieval of the treated formalin-fixed, paraffin-embedded human tissue specimen in the presence of a retrieval solution; co-incubating the formalin-fixed, paraffin-embedded human tissue specimen after the antigen retrieval step with a blocking solution to block nonspecific antigens; mixing the blocked formalin-fixed, paraffin-embedded human tissue specimen with a fixed concentration of an AKR1C3 monoclonal antibody solution for primary antibody incubation; and mixing the primary antibody-incubated formalin-fixed, paraffin-embedded human tissue specimen with a fixed concentration of a secondary antibody solution for secondary antibody incubation. The AKR1C3 detection method can be applied to detect AKR1C3 expression levels in various cancer tumor tissues, and provides stable staining results with good sensitivity, accuracy, and consistency.

Description

[Technical Field]

[0001] The present invention relates to the technology of cancer treatment, and more particularly to a method for detecting AKR1C3, and a diagnostic kit for detecting AKR1C3, and the use of the same. [Background technology]

[0002] Immunohistochemical (IHC) staining has traditionally been an effective method for detecting specific enzymes or proteins in patient pathological tissues. However, existing IHC methods are designed to detect AKR1C3 expression levels and are often developed for only a single type of cancerous tumor tissue. For example, detecting hepatocellular carcinoma requires the development of a separate IHC staining method, and detecting prostate cancer requires the development of yet another IHC staining method. In other words, existing IHC staining detection methods cannot achieve staining and detection of AKR1C3 (aldoketeductase 1C3) expression levels in various cancerous tissues with a single IHC assay.

[0003] Furthermore, since further cancer treatment regimens need to be determined based on IHC detection results, IHC assays must provide stable staining results. In other words, IHC assays used in a wide range of commercial kits need to have good sensitivity, accuracy, and consistency (different laboratories, different operators, different surgical times) and be applicable to various cancer tumor tissues.

[0004] However, the staining results of IHC assays are affected by various factors (Fang Jiedi, Wang Xiaoxing, Zhang Mengling et al., Effect of water quality on immunohistochemical staining results [J]. Journal of Clinical and Experimental Pathology, 2019, 35(04):111-113; Liu Haiyang, Wang Xiaojun, Zhang Haiyu, et al., Effect of microwave heating activation and hydrochloric acid hydrolysis activation methods on immunohistochemical staining results of rat brain tissue [J]. Journal of Ningxia Medical University, 2011(11):1115-1116; Zhang Wei, Liang Yingjie, Effect of different antigen activation methods and staining conditions on P53 protein immunohistochemical results [J]. Chinese Journal of Histochemistry and Cytochemistry, 2002(2); Liu Xianyan, Yang Jian, Chen Ying, et al. al., Effects of different antigen retrieval solutions and retrieval methods on immunohistochemical staining results [J]. Journal of Guangdong Medical College, 2013(05):43-44; Luo Xinlan, Lin Xingtao, Luo Donglan et al., Effects of antigen retrieval solutions containing different components at pH 9.0 on immunohistochemical staining results [J]., Chinese Journal of Pathology, 2012, 41(003):192-194; Cai Guangling, Yu Guangyin, Zhao Yang., Effects of pH of antigen retrieval solution on immunohistochemical staining of lymphoid tissue [J]., Acta Medicinae Sinica, 2005, 18(4):501-502; Du Juan, Shi Xueying, Zheng Jie et al.The effects of pH of antigen retrieval solution and activation time on immunohistochemical staining efficacy are influenced by [J], Journal of Peking University (Health Sciences), 2005, 037(002):195-197; and Meng Kui, Zhou Xiaojun, The role of antigen recovery technology in immunohistochemistry [J], Chinese Journal of Histochemistry and Cytochemistry, 2001, 10(001):109-111.], and the processes affected include the antigen retrieval process and the staining process. Influencing factors and conditions include heating temperature, heating time, water quality used, composition of antigen retrieval solution, pH of antigen retrieval solution, and activation reaction time, and it is certain that differences in tissue type greatly affect the IHC staining detection results. These factors lead to instability in the staining results of IHC assays disclosed in the prior art, i.e., a lack of good sensitivity, accuracy, and consistency, and the IHC assays disclosed in the prior art cannot be applied to the detection of many different types of cancer. Therefore, there is a need to develop an AKR1C3 detection method that is applicable to detecting AKR1C3 expression levels in various cancerous tumor tissues and that provides stable staining results. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the present invention aims to provide an AKR1C3 detection method that can be applied to the detection of AKR1C3 expression levels in various cancer tumor tissues, provides stable staining results, and exhibits good sensitivity, accuracy, and consistency, as well as a diagnostic kit for AKR1C3 detection and its use. [Means for solving the problem]

[0006] Based on the above objectives, one aspect of the present invention is an AKR1C3 detection method in which the AKR1C3 expression level in an isolated formalin-fixed paraffin-embedded human tissue specimen is detected by immunohistochemical staining, a) Antigen retrieval step, in which a formalin-fixed, paraffin-embedded human tissue sample is heated at 90-115°C for 17-30 minutes in the presence of an antigen retrieval solution to activate the antigen, b) Primary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is mixed with an AKR1C3 monoclonal antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, c) Secondary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after the primary antibody incubation step is mixed with a secondary antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, This is an AKR1C3 detection method that includes [specific details omitted].

[0007] In a preferred embodiment of the present invention, in step a) antigen retrieval, the pH of the antigen retrieval solution is 2.0 to 9.0. More preferably, the pH of the antigen retrieval solution is 6.0 to 9.0. More preferably, the pH of the antigen retrieval solution is 6.0.

[0008] In a preferred embodiment of the present invention, in step a) antigen retrieval, the antigen retrieval solution comprises a sodium citrate antigen retrieval solution or an EDTA antigen retrieval solution.

[0009] In a preferred embodiment of the present invention, in step a) antigen retrieval, the formalin-fixed paraffin-embedded human tissue sample is heated at 92-102°C for 18-25 minutes. More preferably, the formalin-fixed paraffin-embedded human tissue sample is heated at 97°C for 20 minutes.

[0010] In a preferred embodiment of the present invention, in step b) primary antibody incubation, the concentration of the AKR1C3 monoclonal antibody solution is 1.0 to 3.0 μg / mL. More preferably, the concentration of the AKR1C3 monoclonal antibody solution is 1.2 μg / mL, and / or In step c) secondary antibody incubation, the concentration of the secondary antibody solution is 1.0 to 3.0 μg / mL. More preferably, the concentration of the secondary antibody solution is 1.2 μg / mL.

[0011] In a preferred embodiment of the present invention, both the AKR1C3 monoclonal antibody solution and the secondary antibody solution are NaN3, H + Cl - and contains tromethamine.

[0012] In a preferred embodiment of the present invention, the AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting them with an antibody dilution buffer, the antibody dilution buffer comprising the following components: A 0.02-0.08 mol / L Tris-HCl buffer containing 0.05-0.15% polyethylene glycol or Tween by mass, and 0.010-0.020 mol / L sodium azide; More preferably, the solution comprises 0.05 mol / L Tris-HCl buffer containing 0.1% by mass polyethylene glycol or Tween and 0.015 mol / L sodium azide.

[0013] In a preferred embodiment of the present invention, in step b) primary antibody incubation, the formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is incubated with the AKR1C3 monoclonal antibody solution for 30 to 45 minutes. More preferably, the formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is incubated with the AKR1C3 monoclonal antibody solution for 45 minutes.

[0014] In a preferred embodiment of the present invention, in the c) secondary antibody incubation step, the formalin-fixed paraffin-embedded human tissue sample after the primary antibody incubation step is incubated with the secondary antibody solution for 30 to 45 minutes. More preferably, the formalin-fixed paraffin-embedded human tissue specimen after the primary antibody incubation step is incubated with the secondary antibody solution for 30 minutes.

[0015] In a preferred embodiment of the present invention, in the step b) of primary antibody incubation, the AKR1C3 monoclonal antibody is a mouse monoclonal antibody, and / or in the step c) of secondary antibody incubation, the secondary antibody is a goat anti-mouse antibody, a rabbit anti-mouse antibody, a horse anti-mouse antibody or a donkey anti-mouse antibody.

[0016] In a preferred embodiment of the present invention, after the step c) of secondary antibody incubation, a step d) of staining and sealing is further included, in which the formalin-fixed paraffin-embedded human tissue specimen is stained with hematoxylin, and the stained specimen is dehydrated and sealed. is further included.

[0017] In a preferred embodiment of the present invention, before the step a) of antigen activation, the formalin-fixed paraffin-embedded human tissue specimen is dewaxed with an organic solvent, and the dewaxed specimen is sequentially washed with alcohols having different water contents and finally washed with water. Preferably, the organic solvent is acetone, toluene or xylene, and more preferably, the organic solvent is xylene, and / or Preferably, the alcohol is ethanol or methanol, and more preferably, the alcohol is ethanol, and / or Preferably, a step a1) of dewaxing and rehydrating is further included, in which the dewaxed specimen is first washed with absolute ethanol and then washed with ethanol having a volume fraction of 90 to 97%.

[0018] In a preferred embodiment of the present invention, between the step a) of antigen activation and the step b) of primary antibody incubation, b1) Blocking of nonspecific antigens: The formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is co-incubated with a blocking solution to block nonspecific antigens. It further includes, Preferably, the blocking solution is the serum of an animal from which the AKR1C3 monoclonal antibody is derived. More preferably, the blocking solution is mouse serum.

[0019] In a preferred embodiment of the present invention, the formalin-fixed, paraffin-embedded human tissue specimen is a breast cancer tissue specimen, a colorectal cancer tissue specimen, an esophageal cancer tissue specimen, a gastric cancer tissue specimen, a hepatocellular carcinoma tissue specimen, a non-small cell lung cancer tissue specimen, a prostate cancer tissue specimen, a renal cell carcinoma specimen, a peripheral T-cell lymphoma specimen, or a nodular NK / T-cell lymphoma specimen.

[0020] Based on the same inventive concept, another aspect of the present invention is: Antigen retrieval solution and A solution of AKR1C3 monoclonal antibody with a concentration of 0.5-5.0 μg / mL, A secondary antibody solution with a concentration of 0.5-5.0 μg / mL, This is a diagnostic kit for detecting AKR1C3, which includes [specific component / method].

[0021] In a preferred embodiment of the present invention, the pH of the antigen retrieval solution is 2.0 to 9.0. More preferably, the pH of the antigen retrieval solution is 6.0 to 9.0. More preferably, the pH of the antigen retrieval solution is 6.0.

[0022] In a preferred embodiment of the present invention, the antigen retrieval solution comprises a sodium citrate antigen retrieval solution or an EDTA antigen retrieval solution.

[0023] In a preferred embodiment of the present invention, the concentration of the AKR1C3 monoclonal antibody solution is 1.0 to 3.0 μg / mL. More preferably, the concentration of the AKR1C3 monoclonal antibody solution is 1.2 μg / mL, and / or The concentration of the secondary antibody solution is 1.0 to 3.0 μg / mL. More preferably, the concentration of the secondary antibody solution is 1.2 μg / mL.

[0024] In a preferred embodiment of the present invention, both the AKR1C3 monoclonal antibody solution and the secondary antibody solution are NaN3, H + Cl - and contains tromethamine.

[0025] In a preferred embodiment of the present invention, the AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting them with an antibody dilution buffer, the antibody dilution buffer comprising the following components: A 0.02-0.08 mol / L Tris-HCl buffer containing 0.05-0.15% polyethylene glycol or Tween by mass, and 0.010-0.020 mol / L sodium azide; more, A 0.05 mol / L Tris-HCl buffer containing 0.1% polyethylene glycol or Tween by mass and 0.015 mol / L sodium azide. Includes.

[0026] In a preferred embodiment of the present invention, the AKR1C3 monoclonal antibody is a mouse monoclonal antibody, and / or The secondary antibody is a goat anti-mouse antibody, rabbit anti-mouse antibody, horse anti-mouse antibody, or donkey anti-mouse antibody.

[0027] In a preferred embodiment of the present invention, the AKR1C3 detection diagnostic kit includes a blocking solution. Preferably, the blocking solution is the serum of the animal from which the AKR1C3 monoclonal antibody is derived. More preferably, the blocking solution is mouse serum.

[0028] In a preferred embodiment of the present invention, the AKR1C3 detection diagnostic kit further comprises Includes a negative control reagent solution and instructions.

[0029] Based on the same inventive concept, another aspect of the present invention is the use of the above-mentioned AKR1C3 detection diagnostic kit in the preparation of agents for the treatment of cancer, tumors, or cell proliferation disorders.

[0030] In a preferred embodiment of the present invention, the above use is: The process involves obtaining the AKR1C3 expression level in formalin-fixed paraffin-embedded human tissue specimens isolated from patients using the aforementioned AKR1C3 detection diagnostic kit, and The method includes the step of administering an AKR1C3-activating anticancer agent to a patient whose AKR1C3 expression level is above a predetermined expression level.

[0031] In preferred embodiments of the present invention, the AKR1C3-activated anticancer agent satisfies at least one of the following definitions, but is not limited to these: A. The inhibitory effect of compounds on cancer cell proliferation detected in the presence of AKR1C3 inhibitors is lower than the inhibitory effect in the absence of AKR1C3 inhibitors. The inhibitory effects of compounds on the proliferation of cancer cells with different levels of AKR1C3 enzyme expression differed significantly; the inhibitory effect on cancer cells with high AKR1C3 enzyme expression was much greater than that on cancer cells with low AKR1C3 enzyme expression. C. If an aldehyde-ketone compound having a carbon-oxygen double bond exhibits significantly different inhibitory effects on the proliferation of cancer cells with different AKR1C3 enzyme expression levels, and the inhibitory effect on the proliferation of cancer cells with high AKR1C3 enzyme expression is far greater than the inhibitory effect on cancer cells with low AKR1C3 enzyme expression, and the difference in inhibitory effects of the corresponding hydroxyl-containing alcohol compound on the proliferation of cancer cells with different AKR1C3 enzyme expression levels is small or equivalent, then the aldehyde-ketone compound having a carbon-oxygen double bond is an AKR1C3 activating anticancer agent, and the corresponding hydroxyl-containing alcohol compound is the parent drug.

[0032] In a preferred embodiment of the present invention, the AKR1C3-activating anticancer agent is a compound with the following structure: [ka] [ka] [ka] [ka] Alternatively, see PCT / NZ2019 / 050030 (International Publication No. 2019 / 190331) for 5-nitrobenzenesulfonamide dibromide, bromomethylsulfonate and bis(methylsulfonyl) mustard (compounds 562-674), 3-methyl-5-nitrobenzenesulfonamide dibromide, bromomethylsulfonate and bis(methylsulfonyl) mustard (compounds 679-791), 3-trifluoromethyl-5-nitrobenzenesulfonamide dibromide, bromomethylsulfonate and bis(methylsulfonyl) mustard. Compounds such as methanesulfonates of fo-nitrobenzenesulfonamide dibromide, bromomethylsulfonate and bis(methylsulfonyl) mustard (compounds 1030-1142), and 5-nitrobenzenesulfonamide bis(methylsulfonyl) mustard (compounds 640.Ms, 641.Ms, 642.Ms, 643.Ms, 644.Ms, 757.Ms, 758.Ms, 991.Ms, 992.Ms, 1108.Ms and 1109.Ms), or [ka] or a pharmaceutically acceptable salt or isomer thereof, Compounds Ex1 to Ex170 in PCT / IB2020 / 057285 (International Publication No. 2021 / 005586), or [ka] or selected from its pharmaceutically acceptable salts or isomers, Preferably, the AKR1C3-activated anticancer agent has the following structure: [ka] or [ka] or [ka] Alternatively, it may be selected from a pharmaceutically acceptable salt or isomer thereof.

[0033] In preferred embodiments of the present invention, the cancer, the tumor, or the cell proliferation disorder is Lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, renal cell carcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial cancer, osteocytic carcinoma, epithelial carcinoma, bile duct cancer, choriocarcinoma, embryonic carcinoma, seminoma, Wilms' tumor, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hemocytoblastoma, vocal cords Neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystomas, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondrosarcoma, myxochondrosarcoma, myxochondrosarcoma, myxoblastoma, liposarcoma, lipoma, lipodenoma, lipoblastoma, lipochondroma, lipofibroma, lipohangoma, myxolipoma, chondrosarcoma, chondromyoma, chordoma, choriocarcinoma, Choriocarcinoma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, bone cyst, bone dentoma, bone fibroma, bone fibrosarcoma, angiosarcoma, hemangioma, angiolipoma, angiochondroma, hemangioblastoma, keratogenic hemangioma, angioglioma, hemangioendothelioma, angiofibroma, angiomyoma, angiomyoma, angiomyoma, angiolymphangioma, angiolipoma, angiolipoma, angiomyolipoma, angiomyoneuroma, angiomyxoma, angioretoma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphomyxoma, lymphosarcoma, This includes lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, peripheral T-cell lymphoma, nodular NK / T-cell lymphoma, endothelioma, endothelial cell tumor, synoviomas, synovial sarcomas, mesothelioma, connective tissue tumors, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyomyoblastoma, leiomyofibrilloma, rhabdomyofibroma, rhabdomyosarcoma, rhabdomyofibroma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic disease cells, polycythemia, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma. Preferably, the cancer, the tumor, or the cell proliferation disorder is Ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma or nodular NK / T-cell lymphoma Includes. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows IHC stained images of CRC samples after activation with a high pH antigen retrieval solution (Scanscope scan: 0.4×, CRC sample: 335933-P). Figure a is 1:1000 and corresponds to antibodies with incubation times of 20 minutes for the primary and secondary antibodies, respectively. Figure b is 1:2000 and corresponds to antibodies with incubation times of 45 minutes for the primary and secondary antibodies, respectively. Figure c is 1:2000 and corresponds to antibodies with incubation times of 60 minutes for the primary and secondary antibodies, respectively. [Figure 2] Figure 2 shows IHC stained images of CRC samples after activation with a low pH antigen retrieval solution (Scanscope scan: 0.4×, CRC sample: 335933-P). Figure a is 1:1000 and corresponds to antibodies with incubation times of 30 minutes for the primary and secondary antibodies, respectively. Figure b is 1:2000 and corresponds to antibodies with incubation times of 45 minutes for the primary and secondary antibodies, respectively. Figure c is 1:2000 and corresponds to antibodies with incubation times of 60 minutes for the primary and secondary antibodies, respectively. [Figure 3] Figure 3 shows photographs of IHC staining results for gastric cancer samples at low pH. Figure a is 1:1000 and corresponds to antibodies with incubation times of 30 minutes for the primary and secondary antibodies, respectively. Figure b is 1:2000 and corresponds to antibodies with incubation times of 45 minutes for the primary and secondary antibodies, respectively. Figure c is 1:2000 and corresponds to antibodies with incubation times of 45 minutes for the primary and secondary antibodies, respectively. [Figure 4]Figure 4 shows photographs of IHC staining results for breast cancer samples at low pH. Figure a is 1:1000 and corresponds to antibodies with primary and secondary antibody incubation times of 30 minutes and 30 minutes, respectively. Figure b is 1:2000 and corresponds to antibodies with primary and secondary antibody incubation times of 45 minutes and 30 minutes, respectively. Figure c is 1:2000 and corresponds to antibodies with primary and secondary antibody incubation times of 45 minutes and 45 minutes, respectively. [Figure 5] Figure 5 shows photographs comparing staining performance under different monoclonal antibody dilutions and incubation times using normal colon tissue:391761-YN as an example (Scanscope scan 4×). Figure a is 1:1000, corresponding to antibodies with primary and secondary antibody incubation times of 30 minutes and 30 minutes, respectively. Figure b is 1:2000, corresponding to antibodies with primary and secondary antibody incubation times of 45 minutes and 30 minutes, respectively. Figure c is 1:2000, corresponding to antibodies with primary and secondary antibody incubation times of 30 minutes and 30 minutes, respectively. [Figure 6] Figure 6 shows a comparison of staining consistency between five different normal colon tissues using the optimal staining protocol (low pH TRS, 97°C, 20 min, AKR1C3 dilution: 1:2000, 30 min, HRP incubation time: 30 min, Scanscope scan 2×). Figure a corresponds to sample 390211-YN, Figure b to sample 390650-YN, Figure c to sample 391182-YN, Figure d to sample 391761-YN, and Figure e to sample 3919951-YN. [Figure 7] Figure 7 shows photographs of AKR1C3 staining in normal tissue using optimal staining conditions. Figures a and b correspond to low-magnification and high-magnification stained images of normal tonsil tissue, respectively; Figures c and d correspond to low-magnification and high-magnification stained images of normal stomach tissue, respectively; and Figures e and f correspond to low-magnification and high-magnification stained images of normal colon tissue, respectively. [Figure 8]Figure 8 shows AKR1C3 stained images of non-small cell lung cancer using an optimal protocol. Figures a and b correspond to low-magnification and high-magnification stained images of sample 1 (F102582A22), respectively, and Figures c and d correspond to low-magnification and high-magnification stained images of sample 2 (F134064A12), respectively. [Figure 9] Figure 9 shows AKR1C3 stained images of gastric cancer using an optimal protocol. Figures a and b correspond to low-magnification and high-magnification stained images of sample 1 (F180723A5), respectively, with upward arrows indicating tumor cells and downward arrows indicating the remaining normal gastric mucosal epithelium. Figures c and d correspond to low-magnification and high-magnification stained images of sample 2 (F180684A3), respectively, with downward arrows indicating tumor cells and upward arrows indicating the remaining normal gastric mucosal epithelium. [Figure 10] Figure 10 shows images of AKR1C3 staining in breast cancer using the optimal protocol. Figures a and b correspond to low-magnification and high-magnification stained images of sample 1 (F162870A5), respectively, and Figures c and d correspond to low-magnification and high-magnification stained images of sample 2 (F130368B3), respectively. [Figure 11] Figure 11 shows AKR1C3 stained images of hepatocellular carcinoma using an optimal protocol. Figures a and b correspond to low-magnification and high-magnification stained images of sample 1 (DLV13050B3), respectively, and Figures c and d correspond to low-magnification and high-magnification stained images of sample 2 (DLV13052B5), respectively. [Figure 12] Figure 12 shows AKR1C3 stained images of colorectal cancer using an optimal protocol. Figures a and b correspond to low-magnification and high-magnification stained images of the sample (335933-P), respectively. [Figure 13]Figure 13 shows photographs of staining in normal colon tissue with both positive and negative components under the optimal protocol for the AKR1C3 IHC assay, scanned at different magnifications. Figure a corresponds to the negative control reagent (Scanscope scan 4×), Figure b corresponds to AKR1C3 (Scanscope scan 10×), Figure c corresponds to AKR1C3 (Scanscope scan 4×), and Figure d corresponds to AKR1C3 (Scanscope scan 10×). [Figure 14] Figure 14 shows photographs of tissue control samples used for each staining procedure, with normal colon tissue containing both positive and negative components serving as controls for double-positive and negative tissues. Figure a corresponds to the negative control reagent (Scanscope 4× scan), and Figure b corresponds to AKR1C3 staining (Scanscope 4× scan). [Figure 15] Figure 15 shows a photograph of sample F151286A5HCC with an H score of 300 (3+:100%), where all tumor cells show strong cytoplasmic / nuclear staining, while normal hepatocytes (arrows) near the cancerous lesion, as well as stromal and endothelial cells, show different staining intensities as internal controls. Figure a corresponds to a lower magnification (Scanscope scan 0.4×), and Figure b corresponds to a higher magnification (Scanscope scan 10×). [Figure 16] Figure 16 shows a photograph of sample F151725A1EC with an H score of 160 (0:0%; 1+:60%; 2+:20%; 3+:20%), where tumor cells show different intensities of cytoplasmic / nuclear staining. Figure a corresponds to a lower magnification (Scanscope scan 0.4×), and Figure b corresponds to a higher magnification (Scanscope scan 10×). [Figure 17] Figure 17 shows a photograph of sample F152459A4GC with an H score of 35 (0: 85%, 1+: 5%, 2+: 0%, 3+: 10%), where tumor cells show different intensities of cytoplasmic / nuclear staining, with the area shown decreasing as the magnification increases (up arrow), and endothelial cells showing staining as an internal control (down arrow). Figure a corresponds to low magnification (Scanscope scan 0.4×), and Figure b corresponds to high magnification (Scanscope scan 10×). [Figure 18]Figure 18 shows a photograph of sample F151653A1CRC with an H score of 120 (0:30%, 1+:30%, 2+:30%, 3+:10%), where tumor cells show different intensities of cytoplasmic / nuclear staining, tumor tissue is indicated by a blue arrow to the right, and normal tissue is indicated by an arrow to the left. Figure a corresponds to a lower magnification (Scanscope scan 0.4×), and Figure b corresponds to a higher magnification (Scanscope scan 10×). [Figure 19] Figure 19 shows a photograph of sample F183410A4PC with an H score of 0 (0:100%), in which none of the tumor cells show cytoplasmic / nuclear staining for AKR1C3, but endothelial cells show staining as an internal control (arrow). Figure a corresponds to a lower magnification (Scanscope scan 0.4×), and Figure b corresponds to a higher magnification (Scanscope scan 10×). [Modes for carrying out the invention]

[0035] Unless otherwise defined, the technical or scientific terms used in one or more examples herein have the ordinary meanings understood by those skilled in the art of the field to which this disclosure belongs.

[0036] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the pharmaceuticals, reagents, and other raw materials used in the following examples are all commercially available products.

[0037] The terms “patient” and “subject” are used interchangeably to refer to mammals requiring treatment for cancer. Generally, a patient is a human. Generally, a patient is a human who has been diagnosed with cancer. In certain embodiments, “patient” or “subject” may refer to non-human mammals, such as non-human primates, dogs, cats, rabbits, pigs, mice, or rats, used for screening, characterizing, and evaluating drugs and treatments.

[0038] A “prodrug” is a compound that, after administration, is metabolized or otherwise converted into a bioactive or more active compound (or drug) with respect to at least one property. Prodrugs are chemically modified to be less active or inactive to a drug, such that the corresponding drug is produced by metabolism or other biological processes after the prodrug is administered. Prodrugs may have altered metabolic stability or transport properties, fewer side effects or lower toxicity, or improved flavor compared to the active drug (see, for example, Nogrady, 1985, Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pages 388-392 (incorporated herein by reference)). Prodrugs may be synthesized using reactants other than the corresponding drug.

[0039] "Treatment" of a patient's condition means taking steps to obtain a beneficial or desired outcome, including clinical results. For the purposes of this invention, beneficial or desired clinical results are not particularly limited, but may include: reduction or improvement of one or more symptoms of cancer; reduction of disease severity; delay or slowing of disease progression; reduction, mitigation, or stabilization of the disease state; or other beneficial outcomes. Treatment of cancer may, in some cases, result in a partial response or stabilization of disease progression.

[0040] "Tumor cells" refer to tumor cells of any appropriate species (e.g., mammals such as mice, dogs, cats, horses, or humans).

[0041] The DNA alkylating agents for anticancer prodrugs targeting AKR1C3 overexpression developed by the applicant include: 1) a DNA alkylating agent corresponding to PCT / US2016 / 021581 (International Publication No. 2016 / 145092) (corresponding to Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)); and 2) a DNA alkylating agent corresponding to PCT / US2016 / 062114 (International Publication No. 2017 / 087428) (Chinese Patent Application No. 2016800446081 (Chinese Patent Application Publication No. 10829) The present invention includes a composition, use, and preparation method of (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramide, corresponding to (Specification 0911), and a nitrobenzyl derivative of an anticancer agent corresponding to PCT / US2016 / 025665 (International Publication No. 2016 / 161342) (Chinese Patent Application No. 2016800200132 (Chinese Patent Application Publication No. 108136214)). The compound in the form of a prodrug is reduced under the catalytic action of AKR1C3 in the intracellular biochemical environment to obtain a cytotoxic toxin, thereby exerting a toxic effect on cancer cells.

[0042] In particular, the S-constituent compound named (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylidene)phosphoramide (also known as OBI-3424, AST-3424, or TH-2870) is represented by CAS number 2097713-69-2 and has the following structure: [ka] Phase I trials are being conducted in the United States and China, respectively.

[0043] Since the above-mentioned drugs are effective only in patients expressing AKR1C3, it is necessary to detect the AKR1C3 expression level in patients. In practical application, it is necessary to determine whether tissue samples from patients meet the conditions for administering the above-mentioned drugs in the three patents (Chinese Patent Application Publication No. 107530556, Chinese Patent Application Publication No. 108290911, and Chinese Patent Application Publication No. 108136214) by reaching a predetermined AKR1C3 expression level, and for this purpose, the IHC assay must produce stable staining results. The inventors of this application attempted to use IHC assays disclosed in the prior art to detect AKR1C3 expression levels in various cancer tumor tissues. Since the existing IHC assays were not IHC staining assays available in large-scale commercial kits, but rather specific IHC assays for specific enzymes or proteins developed by specific hospitals or research institutions targeting specific tissues for specific enzymes or proteins, it was found that the IHC assays disclosed in the prior art did not meet the practical needs. These methods lacked good sensitivity, accuracy, and consistency (due to different laboratories, operators, and handling times) and were not applicable to many IHC assays of different cancer tumor tissues. When staining results are unstable, the measurement of AKR1C3 expression levels is inaccurate, leading to unsatisfactory cancer treatment outcomes.

[0044] Conventional IHC assays cannot be applied to the detection of AKR1C3 expression levels in various cancer tumor tissues and suffer from unstable staining results. To address these problems, the inventors of this invention have attempted to further improve existing IHC assays and have provided an AKR1C3 detection method that can be applied to the detection of AKR1C3 expression levels in various cancer tumor tissues, provides stable staining results, and exhibits good sensitivity, accuracy, and consistency.

[0045] One aspect of the present invention is, A method for detecting AKR1C3 in which the expression level of AKR1C3 in isolated formalin-fixed paraffin-embedded (FFPE) human tissue specimens is detected by immunohistochemical staining, a) Antigen retrieval step, in which a formalin-fixed, paraffin-embedded human tissue sample is heated at 90-115°C for 17-30 minutes in the presence of an antigen retrieval solution to activate the antigen, b) Primary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after antigen retrieval is mixed with an AKR1C3 monoclonal antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, c) Secondary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after the primary antibody incubation step is mixed with a secondary antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, This is an AKR1C3 detection method that includes [specific details omitted].

[0046] The measurement sensitivity results demonstrate that the analytical sensitivity of the IHC assay provided by the present invention for measuring AKR1C3 expression levels in various human cancer tissues has acceptable performance characteristics, showing the expected staining pattern and localization of AKR1C3 in the test samples and demonstrating appropriate performance in controls consistent with the baseline values ​​of the analytical sensitivity study. The measurement accuracy results show 100% consistency in between-batch accuracy (days / between operations, between operators and between instruments) and within-batch accuracy, which indicates that it meets the baseline values ​​of 95% or higher based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells. The measurement consistency results show 100% consistency between pathologists based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells, meeting the baseline values ​​of 90% or higher. Overall, the measurements show acceptable results and demonstrate that it is feasible to use the IHC assay, including the key steps described above, for measuring AKR1C3 expression levels in formalin-fixed paraffin-embedded specimens from various human cancer tissues.

[0047] In this invention, formalin-fixed paraffin-embedded specimens are obtained by preparing the specimen (i.e., treating the tissue specimen with formalin and paraffin to obtain a formalin-fixed paraffin-embedded (FFPE) specimen) and then sectioning the FFPE specimen. A typical thickness for a formalin-fixed paraffin-embedded human tissue specimen is 4 mm.

[0048] The tissue fixation process typically causes protein crosslinking, which often occurs when using formalin fixation due to its chemical properties. Therefore, in the immunohistochemical staining method of the present invention, antigen retrieval is a necessary step before antibody labeling, and the antigen retrieval step is required to re-expose the antigen epitopes to antibody binding. The present invention utilizes the action of the antigen retrieval solution and heat to re-expose these antigens by placing the sample to be activated in the antigen retrieval solution and then heating it; that is, antigen retrieval is achieved by a combination of chemical and thermal action. The general procedure involves placing the sample in the antigen retrieval solution, heating it in an autoclave together with the container, maintaining it at a predetermined temperature for a certain period of time, and then removing it and allowing it to cool naturally. In a preferred embodiment of the present invention, in step a) antigen retrieval, the formalin-fixed paraffin-embedded human tissue sample is heated at 92-102°C for 18-25 minutes, and more preferably, the formalin-fixed paraffin-embedded human tissue sample is heated at 97°C for 20 minutes, to obtain the best effect of good staining effect for various cancer tumor tissue samples. The present invention does not limit the cooling temperature after antigen retrieval; the antigen may be cooled naturally to room temperature or to a specific temperature (e.g., 65°C) before subsequent operations.

[0049] Furthermore, the pH of the antigen retrieval solution affects the antigen retrieval effect, and in combination with the subsequent use of hematoxylin staining, in preferred embodiments of the present invention, the pH of the antigen retrieval solution is 2.0 to 9.0, more preferably 6.0 to 9.0, and even more preferably 6.0. The present invention does not limit the composition of the antigen retrieval solution, and can be used in the present invention as long as its pH meets the requirements. Examples of antigen retrieval solutions include, but are not limited to, sodium citrate antigen retrieval solution and EDTA antigen retrieval solution.

[0050] The concentration of the AKR1C3 monoclonal antibody solution significantly affects the operation of the primary antibody incubation step (b) described above. Experimental measurements showed that an AKR1C3 monoclonal antibody solution with a concentration of 1.0 to 3.0 μg / mL, more preferably 1.2 μg / mL, showed good results, and the dilution ratio of the AKR1C3 monoclonal antibody solution at that time was 1:2000.

[0051] To achieve better results, the AKR1C3 monoclonal antibody solution should consist of the following components: A 0.02-0.08 mol / L Tris-HCl buffer (tromethamine-hydrochloride buffer) containing 0.05-0.15% by mass of polyethylene glycol or Tween and 0.010-0.020 mol / L of sodium azide. It was obtained by diluting with a dilution solution containing [the substance]. More preferably, the antibody dilution buffer consists of the following components: A 0.05 mol / L Tris-HCl buffer containing 0.1% polyethylene glycol or Tween by mass and 0.015 mol / L sodium azide. Includes.

[0052] In immunohistochemical staining, the secondary antibody must be an antigen of the primary antibody species. For example, the primary antibody used to detect the B protein of animal A is the animal C anti-B protein antibody of animal A, and the secondary antibody must be the animal D anti-animal C antibody. The present invention is for detecting human AKR1C3 (protein), and the primary antibody used is a mouse anti-human AKR1C3 monoclonal antibody (i.e., mouse AKR1C3 monoclonal antibody), and the secondary antibody is an anti-mouse antibody of another animal (goat, rabbit, horse, donkey, etc.).

[0053] In a preferred embodiment of the present invention, after the c) secondary antibody incubation step d) Staining and sealing process: Formalin-fixed paraffin-embedded human tissue specimens are stained with hematoxylin, and the stained specimens are dehydrated and sealed. It also includes.

[0054] In a preferred embodiment of the present invention, before the antigen retrieval step a), a1) Dewaxing and rehydration process: The formalin-fixed paraffin-embedded human tissue specimen is dewaxed with an organic solvent, the dewaxed specimen is sequentially washed with alcohols of different water content and finally with water, and the dewaxing and rehydration process is performed to replenish the dried specimen for various subsequent operations. Includes.

[0055] For formalin-fixed, paraffin-embedded human tissue specimens, paraffin embedding is performed, and the presence of paraffin can have a significant impact on subsequent staining processes. Therefore, it is necessary to thoroughly dissolve the paraffin using an appropriate organic solvent. Common organic solvents used to dissolve paraffin without losing the sample include acetone, xylene, and toluene, but xylene is more effective and less toxic. After washing away the paraffin, the residual organic solvent must be washed with an alcohol (methanol or ethanol) dissolved in water; ethanol is usually used. To achieve a better cleaning effect, a gradient elution method is used, that is, the dewaxed sample is first washed with anhydrous ethanol, then with ethanol at a volume fraction of 90-97% (e.g., 95%), and finally with water.

[0056] In a preferred embodiment of the present invention, between the a) antigen retrieval step and the b) primary antibody incubation step, b1) Blocking of nonspecific antigens: The formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is co-incubated with a blocking solution to block nonspecific antigens. It also includes.

[0057] Blocking endogenous enzymes and antibodies in tissues is crucial to minimize background staining and reduce false-positive staining. This is typically achieved by incubating the sample with a specific buffer that can block nonspecific sites to which primary or secondary antibodies may bind. A relatively large number of reagents are used for blocking nonspecific antigens, the main purpose of which is to prevent other proteins, biotin, endogenous enzymes, and other substances (interfering substances) in the sample from interfering with the detection results of AKR1C3 detected in the IHC assay. The use of mouse serum compatible with mouse monoclonal antibodies in this invention can mask all complex interfering substances and facilitates the procedure.

[0058] In a preferred embodiment of the present invention, the AKR1C3 detection method is: a1) Dewaxing and rehydration step, in which the formalin-fixed paraffin-embedded human tissue specimen is dewaxed with an organic solvent, and the dewaxed specimen is sequentially washed with alcohols of different water content and finally washed with water, a) Antigen retrieval step, in which the formalin-fixed paraffin-embedded human tissue specimen is dewaxed and rehydrated, and then heated at 90-115°C for 17-30 minutes in the presence of an antigen retrieval solution to activate the antigen, b1) Blocking of nonspecific antigens, which involves co-incubating the formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step with a blocking solution to block nonspecific antigens, b) Primary antibody incubation step, in which a nonspecific antigen-blocking formalin-fixed paraffin-embedded human tissue sample is mixed with an AKR1C3 monoclonal antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, c) Secondary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after the primary antibody incubation step is mixed with a secondary antibody solution at a concentration of 0.5-5.0 μg / mL and incubated for 25-700 minutes, d) Staining and sealing step, in which the formalin-fixed paraffin-embedded human tissue specimen is stained with hematoxylin, and the specimen is dehydrated and sealed after staining. e) Observation and scoring process: Observe the stained human tissue sample and evaluate the AKR1C3 expression level in the human tissue sample according to the degree of staining observed. By using immunohistochemical staining methods including [specific methods], the expression level of AKR1C3 in isolated formalin-fixed paraffin-embedded (FFPE) human tissue specimens is detected.

[0059] Indeed, c) prior to the operation of the secondary antibody incubation step, a further step of blocking nonspecific antigens can be introduced using the serum of the secondary antibody animal as the blocking solution.

[0060] Using the above conditions, IHC assays were performed on formalin-fixed, paraffin-embedded human tissue samples derived from various cancers (tumors), including breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma, and nodular NK / T-cell lymphoma. All of these samples achieved good staining results, demonstrating that the AKR1C3 IHC assay described above is applicable to various cancerous tumor tissues.

[0061] Based on the same inventive concept, another aspect of the present invention is: Antigen retrieval solution and A solution of AKR1C3 monoclonal antibody with a concentration of 0.5-5.0 μg / mL, This is a diagnostic kit for detecting AKR1C3, containing a secondary antibody solution with a concentration of 0.5 to 5.0 μg / mL.

[0062] In a preferred embodiment of the present invention, the AKR1C3 detection diagnostic kit comprises a blocking solution, preferably the blocking solution being the serum of an animal from which the AKR1C3 monoclonal antibody is derived, and more preferably the blocking solution being mouse serum.

[0063] In a preferred embodiment of the present invention, the AKR1C3 detection diagnostic kit is Negative control reagent solution, This includes a manual documenting the relevant operating procedures.

[0064] The negative control reagent solution is added to more reliably ensure the results of the assay, but it is not essential and may or may not be added. The presence of the negative control reagent in the results facilitates control to confirm whether the staining assay procedure has been performed correctly. Preferably, the negative control reagent (NCR) is a commercially available product from DAKO Corporation: FLEX Negative Control, Mouse, (Link). "Link" means used in conjunction with the Dako Autostainer Link48 fully automated immunohistochemistry system used in the following examples.

[0065] Based on the same inventive concept, another aspect of the present invention is the use of the above-mentioned AKR1C3 detection diagnostic kit in the preparation of agents for the treatment of cancer, tumors, or cell proliferation disorders.

[0066] In a preferred embodiment of the present invention, the above use is The process involves obtaining the AKR1C3 expression level in formalin-fixed, paraffin-embedded human tissue samples isolated from patients using the AKR1C3 detection diagnostic kit described above, and The method includes the step of administering an AKR1C3-activating anticancer agent to a patient whose AKR1C3 expression level is above a predetermined level.

[0067] In this invention, since the expression level of AKR1C3 differs depending on the cancer or tumor, the expression level of AKR1C3 in cancer or tumor tissue specimens suitable for administering a drug containing an AKR1C3-activated anticancer prodrug differs accordingly. For some cancers, high expression is necessary for drug administration, while for others, moderate expression is sufficient. A predetermined expression level of AKR1C3 can be expressed by an H-score, and the predetermined expression level of AKR1C3 corresponding to each cancer type can be obtained by statistical methods.

[0068] In the present invention, the AKR1C3-activated anticancer agent certainly includes an AKR1C3-activated anticancer prodrug; that is, the compound in the form of a prodrug is reduced under the catalytic action of AKR1C3 in the intracellular biochemical environment, ultimately yielding a cytotoxic toxin, thereby exerting a toxic effect on cancer cells.

[0069] Broadly speaking, AKR1C3-activated anticancer agents satisfy at least one of the following conditions, but are not limited to these. A. AKR1C3 inhibitors (such as TH-3021 disclosed in the three patents mentioned above, or compound 36, i.e., Flanagan et al., Bioorganic and Medicinal Chemistry (2014), pp. 962-977) [ka] The inhibitory effect of the compound on cancer cell proliferation detected in the presence of AKR1C3 was lower than the inhibitory effect on cancer cells in the absence of AKR1C3 inhibitors (such as TH-3021 disclosed in the three patents mentioned above), and the inhibitory effect on cancer cell proliferation was IC 50 When quantified using AKR1C3 inhibitors, the detected IC of compounds against specific cancer cell lines is 50 IC in the absence of AKR1C3 inhibitors 50 If the value is greater than this, the compound can be determined to be an AKR1C3-activating anticancer drug (lysis-prodrug). Specifically, lysed prodrugs are described in the following patent documents. PCT / US2016 / 021581 (International Publication No. 2016 / 145092) (corresponding to Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)) PCT / US2016 / 062114 (International Publication No. 2017 / 087428) (corresponding to Chinese Patent Application No. 2016800446081 (Chinese Patent Application Publication No. 108290911)) PCT / US2016 / 025665 (International Publication No. 2016 / 161342) (corresponding to Chinese Patent Application No. 2016800200132 (Chinese Patent Application Publication No. 108136214)) PCT / NZ2019 / 050030 (International Publication No. 2019 / 190331) Compounds disclosed in Chinese Patent Application No. 019800234236 (Chinese Patent Application Publication No. 111918864) The above-mentioned patent documents are incorporated in their entirety herein. PCT / US2016 / 021581 (International Publication No. 2016 / 145092) (corresponding to Chinese Patent Application No. 2016800150788 (Chinese Patent Application Publication No. 107530556)), PCT / US2016 / 062114 (International Publication No. 2017 / 087428) (corresponding to Chinese Patent Application No. 2016800446081 (Chinese Patent Application Publication No. 108290911)) The compounds described in PCT / US2016 / 025665 (International Publication No. 2016 / 161342) (corresponding to the specification of Chinese Patent Application No. 2016800200132 (Chinese Patent Application Publication No. 108136214)) are prodrugs that are metabolized by final dissolution and [Chemical formula] generate not only the active parent drug of, but also paclitaxel, camptothecin and other drugs. The compounds disclosed in PCT / NZ2019 / 050030 (International Publication No. 2019 / 190331) (corresponding to the specification of Chinese Patent Application No. 019800234236 (Chinese Patent Application Publication No. 111918864)) are prodrugs that are metabolized by final dissolution and generate the active parent drug with a nitrogen mustard structure. B. The inhibitory effects of the compounds on the proliferation of cancer cells with different AKR1C3 enzyme expression levels are significantly different. The inhibitory effect on the proliferation of cancer cells with high AKR1C3 enzyme expression is much greater than that on cancer cells with low AKR1C3 enzyme expression. When quantifying the inhibitory effect on the proliferation of cancer cells using IC 50 if the IC 50 of the compound against cancer cells with increasing AKR1C3 enzyme expression level is less than the IC 50 of cancer cells with decreasing AKR1C3 enzyme expression level, the compound can be determined, specifically, it can be determined as the compound disclosed in PCT / CN2020 / 120281 (International Publication No. 2021 / 068952). The above patent documents are incorporated herein in their entirety. C. aldoketeductase 1C3 (AKR1C3) has the ability to reduce certain aldehyde-ketone compounds containing a carbon-oxygen double bond to corresponding alcohol compounds containing a hydroxyl group. If the inhibitory effect of certain aldehyde-ketone compounds containing a carbon-oxygen double bond on the proliferation of cancer cells differs significantly depending on the expression level of the AKR1C3 enzyme, and the inhibitory effect on the proliferation of cancer cells with a high expression level of the AKR1C3 enzyme is very large, then it is larger than that on cancer cells with a low expression level of the AKR1C3 enzyme, and if the difference in the inhibitory effect of the corresponding alcohol compound containing a hydroxyl group on the proliferation of cancer cells with different expression levels of the AKR1C3 enzyme is small or equivalent, then the aldehyde-ketone compound containing a carbon-oxygen double bond is an AKR1C3-activating anticancer agent (reducing prodrug), and the corresponding alcohol compound containing a hydroxyl group is the parent drug (drug), specifically the compound disclosed in PCT / IB2020 / 057285 (International Publication No. 2021 / 005586). The aforementioned patent documents are incorporated in their entirety herein.

[0070] All compounds of the general formula and specific compounds disclosed in the above patent application belong to the category of AKR1C3-activated anticancer agents / prodrugs, and the above application incorporates all of its disclosures herein.

[0071] Furthermore, compounds with the following structures are preferred, and all of them are AKR1C3-activating anticancer agents / prodrugs: [ka] [ka] [ka] [ka] [ka] The above-mentioned A-type AKR1C3-activating anticancer agent, or [ka] Alternatively, a pharmaceutically acceptable salt or isomer thereof, which is the above-mentioned B-type AKR1C3 activating anticancer agent, or [ka] Alternatively, a pharmaceutically acceptable salt or isomer thereof, which is the above-mentioned C-type AKR1C3 activating anticancer agent.

[0072] In this invention, a total of 46 tissue samples from nine markers were used, including 5 RCC (renal cell carcinoma) samples, 5 HCC (hepatocellular carcinoma) samples, 5 NSCLC (non-small cell lung cancer) samples, 5 GC (gastric cancer) samples, 5 PC (prostate cancer) samples, 5 EC (esophageal cancer) samples, 5 CRC (colorectal cancer) samples, 6 peripheral T-cell lymphoma samples, and 5 NK / T-cell lymphoma samples.

[0073] For each staining procedure, normal colon tissue containing both positive and negative components was used as a control for double-positive and double-negative tissues.

[0074] All of the above samples were formalin-fixed, paraffin-embedded (FFPE) human tissue specimens, cut into 4 μm thick tissue samples, placed on positively charged slides, and stored at ambient temperature until stained.

[0075] The biochemical and chemical reagents used for the experiment are listed in Table 1 below.

[0076] [Table 1-1]

[0077] [Table 1-2]

[0078] equipment Ventana Benchmark Ultra fully automated immunohistochemistry system (also known as a tissue stainer, serial numbers: 311434, 316829)

[0079] Dako Autostainer Link 48 Fully Automated Immunohistochemical Staining System (also known as a tissue sample stainer, serial numbers: AS5085D1611, AS2370D1203)

[0080] DAKO PT Link Fully Automated Immunohistochemistry Sample Preparation System (Serial Numbers: MY1716P184, PT3543Y1310)

[0081] Sakura Tissue-Tek DRS Slide Staining Kit (Serial Number: 49310219-0409)

[0082] Sakura Slide Heater (Serial Number: 14881799-0409)

[0083] Sakura Slicer (Serial Number: 1429-1407)

[0084] Nikon microscope (Serial number: 940776)

[0085] HANNA pH meter (Serial number: 08678109)

[0086] Positively charged microscope slide

[0087] Aperio Scanscope XT Digital Specimen Scanning System (Serial Number: SS001403)

[0088] The technical solutions of the present invention will be further described below in combination with specific examples. The following examples are intended to illustrate the present invention only and do not limit the scope of protection of the present invention.

[0089] The criteria for evaluating the staining results in the following examples were as follows:

[0090] (1) Explanation of stain Cells labeled with the AKR1C3 antibody show cytoplasmic and / or nuclear staining.

[0091] (2) General scoring guidelines The interpretation of staining or lack thereof was made by morphological studies using appropriate controls, evaluated by qualified pathologists (GH and KZ), and the localization, distribution, and intensity of AKR1C3 staining in tumor specimens were measured.

[0092] For reactivity evaluation, Cellular localization of staining, The intensity of the dyeing, Intracellular localization and, The percentage of cell staining in the target region, This includes the aspect of...

[0093] AKR1C3 measurements were evaluated on a semi-quantitative scale, and the percentage of cell staining was recorded for cytoplasmic and nuclear staining at the following four levels (0, 1+, 2+, and 3+).

[0094] (3) Criteria for scoring tumor samples H-score usage: The degree of staining (i.e., level of AKR1C3 enzyme expression) was scored using the percentage of nuclear-cytoplasmic stained tumor cells (the total value from 0 to 3+ must not exceed 100). 0 (unstained): Values ​​from 0 to 100 Tumor cell nucleus-cytoplasm 1+ (weak staining): Values ​​from 0 to 100 Tumor cell nucleus-cytoplasm 2+ (moderate staining): values ​​from 0 to 100 Tumor cell nucleus-cytoplasm 3+ (strong staining): Values ​​from 0 to 100 Total percentage of nuclear-cytoplasmic positive staining: values ​​from 0 to 100

[0095] The total H score is calculated based on the proportional tumor score for each intensity. The final H score was calculated as follows: H score = (% weak [1+] × 1) + (% medium [2+] × 2) + (% strong [3+] × 3)

[0096] There are no specific cutoff values ​​assigned to the clinical interpretation of AKR1C3 positive and negative states during this validation, and these may be determined after the completion of the clinical trial.

[0097] A total positive % score of ±10% was defined as consistency among pathologists for the same sample. However, if pathologists score the same case as 0% and 1%, this should be considered a contradiction.

[0098] Example 1: Establishment of a method for detecting AKR1C3 In this embodiment, existing methods provided by the commercially available Dako Autostainer Link 48 platform and Ventana Benchmark Ultra platform were first pre-validated. The pre-validation revealed that neither of the existing methods provided by the two platforms could be applied to the AKR1C3 IHC method. Therefore, the conditions for Ventana Benchmark Ultra and Dako Autostainer Link 48 need to be optimized.

[0099] Based on the principles of IHC, and combined with the staining results of prior verification, the inventors of this application determined that the conditions to be optimized are as follows: Optimization of antibody concentration and incubation time, Optimization of antigen retrieval conditions, It was decided that this would include optimizing the conditions of the detection system.

[0100] 1.1 Optimization of antibody concentration Preliminary validation revealed that the background staining of AKR1C3 at 1:100 and 1:500 was extremely strong. The staining at 1:4000 was too weak. In the next step, we focused on AKR1C3 at 1:1000 and 1:2000, but both showed background staining.

[0101] 1.2 Optimization of antigen retrieval conditions Table 2 shows the results of staining tests performed using a high-pH antigen retrieval solution (pH 9.0):

[0102] [Table 2]

[0103] Table 3 shows the results of staining tests performed using a low-pH antigen retrieval solution (pH 6.0):

[0104] [Table 3]

[0105] High pH antigen retrieval solutions resulted in extremely strong background staining, but lower pH significantly reduced background staining. A low pH antigen retrieval solution was used for this verification (Scanscope 1× scan).

[0106] 1.3 Optimal testing of further samples Using a low-pH antigen retrieval solution, optimal testing was performed on further samples, including normal tissue and various solid tumors, to determine the optimal antibody concentration and incubation time.

[0107] [Table 4]

[0108] As can be seen from the staining results in Figures 3a-3c, background / nonspecific staining is observed in Figure 3a, appropriate staining is observed in Figure 3b, and background / nonspecific staining is observed in Figure 3c.

[0109] [Table 5]

[0110] As can be seen from the staining results in Figures 4a-4c, background / nonspecific staining is observed in Figure 4a, appropriate staining is observed in Figure 4b, and background / nonspecific staining is observed in Figure 4c.

[0111] Optimal staining performance was determined to have been demonstrated under the following conditions: low pH antigen retrieval solution, 97°C for 20 minutes; AKR1C3 dilution: 1:2000, incubation time: 45 minutes; HRP (horseradish peroxidase) incubation time: 30 minutes. These were measured by staining of positive and negative tissue components, as well as specific positive staining for cell localization and staining intensity range, and showed an optimal signal-to-noise ratio. This optimal staining protocol will be used for validation (Scanscope scan 1×).

[0112] 1.4 QC (Control) Verification Five normal colon tissue samples were pre-validated using the optimal staining protocol described above to test their staining performance and verify whether they were suitable quality control controls for the study.

[0113] [Table 6]

[0114] As can be seen from the staining results in Figures 5a-5c, background / nonspecific staining was observed in Figure 5a, appropriate staining in Figure 5b, and weakly specific staining in Figure 5c. Using the optimal staining protocol, all five normal colon tissues showed consistent optimal staining performance (low pH antigen retrieval solution, 97°C for 20 minutes; 1:2000 dilution with AKR1C3, incubation time: 45 minutes; HRP incubation time: 30 minutes), as shown in Figure 6. These were measured by staining of positive and negative tissue components as well as specific positive staining for cell localization and staining intensity range, showing an optimal signal-to-noise ratio. Next, normal colon tissue containing both positive and negative components was used as a control for bi-positive and bi-negative tissues in each staining procedure of the investigation.

[0115] The optimal staining conditions were determined based on the results of these tests and are shown in Table 7.

[0116] [Table 7]

[0117] In other words, the final method for detecting AKR1C3 is: The antigen retrieval process involves heating a formalin-fixed, paraffin-embedded human tissue sample at 97°C for 20 minutes in the presence of an antigen retrieval solution with a pH of 6.0 to activate the antigen. A primary antibody incubation step comprising mixing a formalin-fixed, paraffin-embedded human tissue sample with a 1.2 μg / mL AKR1C3 monoclonal antibody solution and a negative control reagent solution, and incubating for 45 minutes, wherein the negative control reagent solution is a commercially available product from DAKO Corporation: FLEX Negative Control, Mouse, (Link), The method includes a secondary antibody incubation step in which a formalin-fixed, paraffin-embedded human tissue sample is mixed with a 1.2 μg / mL secondary antibody solution and incubated for 30 minutes.

[0118] IHC staining was performed using optimal staining conditions for normal tonsil tissue, normal gastric tissue, normal colon tissue, non-small cell lung cancer, gastric cancer, breast cancer, hepatocellular carcinoma, and colorectal cancer, as shown in Figures 7-12. Staining of various normal tissues and solid tumors showed optimal signal-to-noise ratios, which were measured by staining of positive and negative tissue components, as well as specific positive staining for cell localization and staining intensity range. Normal tissues: Stromal cells and endothelial cells may exhibit different staining intensity levels.

[0119] 1.5 QC Measurement Normal colon tissue containing both positive and negative components will be used as controls for double-positive and negative tissues in each staining procedure and will be used for the following validation procedures and future in vivo studies.

[0120] As shown in Figure 13, staining tests were performed on normal colon tissue containing positive and negative components using the optimal protocol described above, and the images were scanned simultaneously at different magnifications. The clear distinction between positive and negative staining and the excellent staining effect made it convenient for pathologists to subsequently score and measure the level of AKR1C3 expression.

[0121] Example 2 Sensitivity of the analytical method 2.1 Test Method The sensitivity of this IHC assay was evaluated by staining a total of 46 tissue samples, including 5 RCC, 5 HCC, 5 NSCLC, 5 GC, 5 PC, 5 EC, 5 CRC, 6 peripheral T-cell lymphoma tissue samples, and 5 NK / T-cell lymphoma tissue samples, with AKR1C3 antibody.

[0122] Each staining procedure includes normal colon tissue used as a control for double-positive and double-negative tissues, as well as a negative control reagent (NRC) for each sample. These must first be evaluated by a QBEJ pathologist to show the expected acceptable staining for the biomarker.

[0123] Reference value: (1) Samples stained with NRC must have a specific staining of 0 and a background staining intensity of less than 1+. (2) Samples stained with AKR1C3 antibody must have a nonspecific background staining intensity of less than 1+. (3) Cells stained with AKR1C3 antibody must show appropriate cellular localization.

[0124] 2.2 Test Results The results of the sensitivity evaluation of 46 tissue samples are summarized in Table 8.

[0125] We initially evaluated all results for positive and negative controls for each procedure, as well as controls including negative control reagents for each sample, and they showed staining within the expected acceptable range for the biomarkers.

[0126] [Table 8-1]

[0127] [Table 8-2]

[0128] The results of the analytical sensitivity operations on all control slides and tumor samples demonstrated that the AKR1C3 antibody showed expected staining and localization of AKR1C3 in control and tumor samples of normal colon tissue, according to the reference values ​​described in Example 3.

[0129] Example 3: Accuracy of the analytical method 3.1 Test Method Using five samples with different AKR1C3 expression levels from the above analytical sensitivity study, two operators performed three staining operations using two instruments over three non-consecutive days, and we evaluated the inter-batch (day / operation, operator, and instrument) and intra-batch precision. The precision study plan is shown in Table 9 below.

[0130] The batch-to-batch precision is as follows: (1) Day-to-day / batch precision: The staining process needs to be carried out in a cycle of three non-consecutive days. One NRC section and one AKR1C3 sample per sample. (2) Accuracy between operators and between instruments: The process requires the use of two staining platforms and the involvement of two operators during the three staining steps. (3) Intra-batch accuracy For batch accuracy, four consecutive samples were required in a single operation. One NRC section and three AKR1C3 samples per sample.

[0131] [Table 9]

[0132] Each staining procedure includes normal colon tissue used as a control for double-positive and negative tissues, as well as a negative control reagent (NRC) for each sample, which must first be evaluated by a pathologist to show the expected acceptable staining for the biomarker. The interpretation of results and scoring criteria are described in detail in "Scoring Criteria for Tumor Samples."

[0133] Reference value: The threshold for inter-batch and intra-batch consistency was maintained at over 95% based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells, as detailed in the "Tumor Sample Scoring Criteria."

[0134] 3.2 Test Results Using five samples with different AKR1C3 expression levels from the analytical sensitivity study described above, two operators performed three staining operations using two instruments over three non-consecutive days. Inter-batch (day / operational, operator-to-operator, and instrument-to-instrument) and intra-batch accuracy were evaluated. The results are summarized in Table 10.

[0135] [Table 10-1]

[0136] [Table 10-2]

[0137] The accuracy of the AKR1C3 IHC assay, performed by two operators using two instruments on five samples with different AKR1C3 expression levels, involved three staining operations over three non-consecutive days, and the results were reproducible. Based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells as detailed above, 100% consistency was observed between and within the operations, which meets the standard of over 95%.

[0138] Example 4: Consistency of the analytical method 4.1 Test Method Two pathologists (GH and KZ) independently evaluated the 46 tissue samples used in the analytical sensitivity study. The interpretation of the results and the scoring criteria are described in detail in "Scoring Criteria for Tumor Samples."

[0139] Reference value: The threshold for interpathologist consistency was 90% or higher, based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells, as detailed in the "Criteria for Scoring Tumor Samples."

[0140] 4.2 Test Results Two pathologists (GH and KZ) independently evaluated the 46 tissue samples used in the analytical sensitivity study. The results are summarized in Table 11.

[0141] [Table 11-1]

[0142] [Table 11-2]

[0143] [Table 11-3]

[0144] The consistency of IHC assays for AKR1C3 between two pathologists (GH and KZ) was 100% consistent (46 / 46) based on AKR1C3 expression in the cytoplasm and nucleus of tumor cells, meeting the reference value of over 90%.

[0145] To illustrate the performance characteristics of this method and these criteria, representative figures of IHC results for a subset of assay samples are shown in Figures 14–19 (original magnification 20x).

[0146] Example 5: Diagnostic kit for detecting AKR1C3 In this embodiment, the AKR1C3 detection diagnostic kit (Kit) is: An antigen retrieval solution with a pH of 6.0, NaN3, H + Cl - and a 1.2 ug / ml AKR1C3 monoclonal antibody solution containing tromethamine, NaN3, H + Cl - and a secondary antibody solution containing tromethamine at a concentration of 1.2 ug / ml, Blocking solution: mouse serum and Negative control reagent solution, This includes a manual documenting the relevant operating procedures.

[0147] A mouse AKR1C3 monoclonal antibody solution at a concentration of 1.2 ug / ml and a secondary antibody solution at a concentration of 1.2 ug / ml were obtained by diluting the antibody dilution solution. The antibody dilution solution contains 0.1% by mass polyethylene glycol or Tween and 0.015 mol / L Tris-HCl buffer (tromethamine-hydrochloride buffer) containing 0.05 mol / L sodium azide. The secondary antibody is a goat anti-mouse antibody, a rabbit anti-mouse antibody, a horse anti-mouse antibody, or a donkey anti-mouse antibody.

[0148] The negative control reagent solution is a commercially available product from DAKO Corporation: FLEX Negative Control, Mouse, (Link).

[0149] Diagnostic kits for detecting AKR1C3 in combination with AKR1C3-activated anticancer prodrugs were typically used for patient screening. These kits allow medical staff to perform detections in different laboratories using standard operating procedures (SOPs) for the detection kits before deciding on drug administration to patients. This ensures that AKR1C3 detection results obtained using the same reagents and procedures match the recommended detection results for specific cancers as described in the drug information leaflet for the AKR1C3-activated anticancer prodrug.

[0150] The specific operating procedures for the kit are described in the instruction manual; that is, the specific operating conditions are described in the instruction manual. Depending on the case, or in a preferred embodiment, the score values ​​of the IHC staining assay using AKR1C3-activated anticancer prodrugs for different types of cancer (tumors) are also described in these instructions. For example, for gastric cancer, it has been statistically determined that the score of the IHC staining detection method using AKR1C3-activated anticancer prodrugs in gastric cancer patients should not be 165 or less. If the score (e.g., H score) of a patient's gastric cancer tissue sample detected and scored using the above kit is 209, the physician can prescribe AKR1C3-activated anticancer prodrugs to this patient. As another example, for esophageal cancer, it has been statistically determined that the score of the IHC staining detection method using AKR1C3-activated anticancer prodrugs in esophageal cancer patients should not be 115 or less. If the score of a patient's esophageal cancer tissue sample detected and scored using the above kit is 105, the physician cannot prescribe AKR1C3-activated anticancer prodrugs to this patient.

[0151] Example 6: Use of AKR1C3 detection method and AKR1C3 detection diagnostic kit in the treatment of cancer, tumors, or cell proliferation disorders. The score value (e.g., H score) of formalin-fixed paraffin-embedded human tissue specimens isolated from gastric cancer patients detected by the AKR1C3 detection method established in Example 1 or the AKR1C3 detection diagnostic kit of Example 5 was 209, which was greater than the predetermined score value of 165. This gastric cancer patient was administered an AKR1C3-activating anticancer drug.

[0152] As verified by existing tests, the following structure [ka] or [ka] Alternatively, AKR1C3-activated anticancer agents selected from their pharmaceutically acceptable salts or isomers may have the best therapeutic effect.

Claims

1. An AKR1C3 detection method is provided, in which the AKR1C3 expression level in isolated formalin-fixed paraffin-embedded human tissue specimens is detected by immunohistochemical staining, a) Antigen retrieval step, in which a formalin-fixed, paraffin-embedded human tissue sample is heated at 92-102°C for 18-25 minutes in the presence of an antigen retrieval solution to activate the antigen, b) Primary antibody incubation step, in which a formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is mixed with an AKR1C3 monoclonal antibody solution at a concentration of 1.0–3.0 μg / mL and incubated for 30–45 minutes, c) Secondary antibody incubation step, in which the formalin-fixed paraffin-embedded human tissue sample after the primary antibody incubation step is mixed with a secondary antibody solution at a concentration of 1.0 to 3.0 μg / mL and incubated for 30 to 45 minutes, Includes, In step a) the antigen retrieval step, the antigen retrieval solution comprises a sodium citrate antigen retrieval solution or an EDTA antigen retrieval solution, and the pH of the antigen retrieval solution is 6.0 to 9.

0. The AKR1C3 monoclonal antibody solution and the secondary antibody solution both contain NaN3, H+, Cl-, and tromethamine. A method for detecting AKR1C3, wherein the formalin-fixed, paraffin-embedded human tissue sample is a breast cancer tissue sample, a colorectal cancer tissue sample, an esophageal cancer tissue sample, a gastric cancer tissue sample, a hepatocellular carcinoma tissue sample, a non-small cell lung cancer tissue sample, a prostate cancer tissue sample, a renal cell carcinoma sample, a peripheral T-cell lymphoma sample, or a nodular NK / T-cell lymphoma sample.

2. The AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting them with antibody dilution buffer, the antibody dilution buffer comprising the following components: A 0.02–0.08 mol / L Tris-HCl buffer containing 0.05–0.15% by mass polyethylene glycol or Tween, and 0.010–0.020 mol / L sodium azide. The AKR1C3 detection method according to claim 1, including the method described in claim 1.

3. b) In the primary antibody incubation step, the AKR1C3 monoclonal antibody is a mouse monoclonal antibody, and / or The AKR1C3 detection method according to claim 1 or 2, wherein in the secondary antibody incubation step c) the secondary antibody is a goat anti-mouse antibody, a rabbit anti-mouse antibody, a horse anti-mouse antibody, or a donkey anti-mouse antibody.

4. c) After the secondary antibody incubation step, d) Staining and sealing process: Stain formalin-fixed paraffin-embedded human tissue specimens with hematoxylin, and then dehydrate and seal the specimens after staining. A method for detecting AKR1C3 according to any one of claims 1 to 3, further comprising:

5. a) Before the antigen retrieval step, The formalin-fixed paraffin-embedded human tissue specimen is dewaxed with an organic solvent, and the dewaxed specimen is sequentially washed with alcohols of different water content, and finally washed with water. The organic solvent is acetone, toluene, or xylene, and / or The alcohol is ethanol or methanol, and / or a1) Dewaxing and rehydration process: First, wash the dewaxed sample with anhydrous ethanol, and then wash it with ethanol at a volume fraction of 90-97%. A method for detecting AKR1C3 according to any one of claims 1 to 4, further comprising:

6. Between the a) antigen retrieval step and the b) primary antibody incubation step, b1) Blocking of nonspecific antigens: The formalin-fixed paraffin-embedded human tissue sample after the antigen retrieval step is co-incubated with a blocking solution to block nonspecific antigens. It further includes, The method for detecting AKR1C3 according to any one of claims 1 to 5, wherein the blocking solution is the serum of an animal from which the AKR1C3 monoclonal antibody is derived.

7. Antigen retrieval solution and A solution of AKR1C3 monoclonal antibody at a concentration of 1.0–3.0 μg / mL, A secondary antibody solution with a concentration of 1.0–3.0 μg / mL, Includes, The pH of the antigen retrieval solution is 6.0 to 9.0, and / or The antigen retrieval solution comprises a sodium citrate antigen retrieval solution or an EDTA antigen retrieval solution. The AKR1C3 monoclonal antibody solution and the secondary antibody solution are both AKR1C3 detection diagnostic kits containing NaN3, H+, Cl-, and tromethamine.

8. The AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting them with antibody dilution buffer, the antibody dilution buffer comprising the following components: A 0.02–0.08 mol / L Tris-HCl buffer containing 0.05–0.15% by mass of polyethylene glycol or Tween, and 0.010–0.020 mol / L of sodium azide; A diagnostic kit for detecting AKR1C3 according to claim 7, comprising:

9. The AKR1C3 monoclonal antibody is a mouse monoclonal antibody, and / or The AKR1C3 detection diagnostic kit according to claim 7 or 8, wherein the secondary antibody is a goat anti-mouse antibody, a rabbit anti-mouse antibody, a horse anti-mouse antibody, or a donkey anti-mouse antibody.

10. Contains a blocking solution, The AKR1C3 detection diagnostic kit according to any one of claims 7 to 9, wherein the blocking solution is the serum of an animal from which the AKR1C3 monoclonal antibody is derived.

11. Negative control reagent solution, Instructions and A diagnostic kit for detecting AKR1C3 according to any one of claims 7 to 10.

12. A diagnostic kit for detecting AKR1C3 according to any one of claims 7 to 11, used for detecting AKR1C3 expression levels in cancer, tumor, or cell proliferation disease tissue.

13. The process involves obtaining the AKR1C3 expression level in formalin-fixed, paraffin-embedded human tissue samples isolated from a patient using the aforementioned AKR1C3 detection diagnostic kit, The process involves administering an AKR1C3-activating anticancer agent to a patient whose AKR1C3 expression level is above a predetermined level, A diagnostic kit for detecting AKR1C3 for use according to claim 12, comprising:

14. The AKR1C3-activating anticancer agent is the AKR1C3 detection diagnostic kit for use according to claim 13, wherein the AKR1C3-activating anticancer agent satisfies at least one of the following definitions: A. The inhibitory effect of a compound on the proliferation of cancer cells detected in the presence of an AKR1C3 inhibitor is lower than the inhibitory effect in the absence of the AKR1C3 inhibitor; B. The inhibitory effects of compounds on the proliferation of cancer cells differed significantly depending on the expression level of the AKR1C3 enzyme; the inhibitory effect on cancer cells with high AKR1C3 enzyme expression was much greater than the inhibitory effect on cancer cells with low AKR1C3 enzyme expression. C. If an aldehyde-ketone compound having a carbon-oxygen double bond exhibits significantly different inhibitory effects on the proliferation of cancer cells with different AKR1C3 enzyme expression levels, and the inhibitory effect on the proliferation of cancer cells with high AKR1C3 enzyme expression is far greater than the inhibitory effect on cancer cells with low AKR1C3 enzyme expression, and the difference in inhibitory effects of the corresponding hydroxyl-containing alcohol compound on the proliferation of cancer cells with different AKR1C3 enzyme expression levels is small or equivalent, then the aldehyde-ketone compound having a carbon-oxygen double bond is an AKR1C3 activating anticancer agent, and the corresponding hydroxyl-containing alcohol compound is the parent drug.

15. The aforementioned AKR1C3-activating anticancer agent is a compound having the following structure: 【Chemistry 1-1】 [Chemistry 1-2] [Chemistry 1-3] [Chemistry 1-4] or [Chemistry 1-5] or a pharmaceutically acceptable salt or isomer thereof, [Chemistry 1-6] A diagnostic kit for detecting AKR1C3 for use according to claim 13 or 14, or selected from a pharmaceutically acceptable salt or isomer thereof.

16. The aforementioned AKR1C3-activated anticancer agent has the following structure: [Chemistry 1-7] or [Chemistry 1-8] or [Chemistry 1-9] A diagnostic kit for detecting AKR1C3 for use according to claim 15, selected from a pharmaceutically acceptable salt or isomer thereof.

17. The aforementioned cancer, the aforementioned tumor, or the aforementioned cell proliferative disorder Lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, renal cell carcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial cancer, osteocytic carcinoma, epithelial carcinoma, bile duct cancer, choriocarcinoma, embryonic carcinoma, seminoma, Wilms' tumor, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hemocytoblastoma, vocal cord neuroma, meningioma, nerve Blastoma, neuroblastoma optica, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroadenoma, fibrochondroma, fibrocystomas, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondrosarcoma, myxochondrosarcoma, myxochondrosarcoma, myxoblastoma, liposarcoma, lipoma, lipodenoma, lipoblastoma, lipochondroma, lipofibroma, lipohangoma, myxolipoma, chondrosarcoma, chondromoma, chondromyoma, chordoma, choriocarcinoma, choriocarcinoma, chorioblastoma, osteosarcoma, osteoblastoma Cell tumor, osteochondrofibroma, osteochondrosarcoma, osteochondroma, bone cyst, bone dentoma, bone fibroma, bone fibrosarcoma, angiosarcoma, hemangioma, angiolipoma, angiochondroma, hemangioblastoma, keratogenic hemangioma, angioglioma, hemangioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipoma, angiomyolipoma, angiomyoneuroma, angiomyxoma, angioretinoma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, peripheral A diagnostic kit for detecting AKR1C3 for use according to any one of claims 12 to 16, comprising T-cell lymphoma, nodular NK / T-cell lymphoma, endothelioma, endothelial cell tumor, synoviomas, synovial sarcomas, mesothelioma, connective tissue tumors, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyomyoblastoma, leiomyomyofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic disease cells, polycythemia, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma.

18. The aforementioned cancer, the aforementioned tumor, or the aforementioned cell proliferative disorder Ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma or nodular NK / T-cell lymphoma A diagnostic kit for detecting AKR1C3 for use according to claim 17, comprising:

Images