Drug or drug composition for treating TFE3 gene rearrangement renal cell carcinoma and use thereof

Abstract

The present invention provides a drug or a drug composition for treating TFE3 gene rearrangement renal cell carcinoma and its use, and relates to the field of drug treatment and application technology of clinical tumors. The drug or drug composition for treating TFE3 gene rearrangement renal cell carcinoma includes sertindole. The drug or drug composition of the present invention can effectively inhibit the proliferation, migration and invasion of TFE3 gene rearrangement renal cell carcinoma, and is expected to be applied to the treatment of clinical TFE3 gene rearrangement renal cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of drug treatment and application of clinical tumors, and in particular to a drug or drug composition for treating TFE3 gene rearrangement renal cell carcinoma and its use. Background Art

[0002] TFE3-rearranged renal cell carcinoma (TFE3-RCC) is a rare subtype of renal cancer. It was first reported in 1986. Because the TFE3 gene in the first band and second subband of the X chromosome of the patient is translocated and forms a fusion gene, it is also called Xp11.2 translocation / TFE3 gene fusion-related renal cell carcinoma (Xp11.2 translocation renal cell carcinoma, Xp11.2 tRCC). In 2004, the WHO classification of renal tumors officially listed it as an independent subtype of renal cancer. Because the TFE3 gene belongs to the microphthalmia transcription factor family (MiTF), Xp11.2 tRCC was classified as MiTF translocation renal cell carcinoma in 2016. The fifth edition of the WHO classification of renal tumors in 2022 renamed Xp11.2 tRCC as TFE3 tRCC and classified it as renal cell carcinoma diagnosed based on molecular technology.

[0003] Currently, there is a lack of universal and effective treatment for TFE3-RCC patients. For localized early renal cancer, the treatment is mainly based on nephron-preserving surgery. Radical nephrectomy can be used for patients with larger tumors. However, there is no effective treatment for patients with local infiltration or distant metastasis who are not suitable for surgery.

[0004] Currently, the efficacy of various targeted therapies such as sunitinib, everolimus, sorafenib, crizotinib, pazopanib and anti-PD-L1 antibody therapy for TFE3-RCC patients has been explored clinically, but due to the small number of samples and lack of systematicity, there is no effective treatment evaluation. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a drug or a drug composition and use thereof for treating TFE3 gene rearrangement renal cell carcinoma. The technical scheme is as follows:

[0006] A medicine or a medicine composition for treating TFE3 gene rearrangement renal cell carcinoma, comprising sertindole.

[0007] Optionally, the medicine or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0008] Optionally, the carrier or excipient includes one or more of a binder, a filler, a diluent, a tabletting agent, a lubricant, a disintegrant, a colorant, and a wetting agent.

[0009] Optionally, the drug or pharmaceutical composition is in the form of tablets, capsules, oral liquids, lozenges, granules, pills, powders, granules, ointments, suspensions, powders, solutions, injections, creams, sprays, drops or patches.

[0010] Optionally, the drug or pharmaceutical composition is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.

[0011] Use of sertindole in preparing a drug for treating TFE3 gene rearrangement renal cell carcinoma.

[0012] Optionally, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0013] Optionally, the carrier or excipient includes one or more of a binder, a filler, a diluent, a tabletting agent, a lubricant, a disintegrant, a colorant, and a wetting agent.

[0014] Optionally, the drug is in the form of tablets, capsules, oral liquids, lozenges, granules, pills, powders, granules, ointments, suspensions, powders, solutions, injections, creams, sprays, drops or patches.

[0015] Optionally, the drug is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.

[0016] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0017] Sertindole (Chinese name: Sertindole) contained in the drug or drug composition of the present invention can effectively inhibit the proliferation, migration and invasion of TFE3 gene rearrangement renal cell carcinoma, with a maximum inhibition rate of 99.99%. When the concentration reaches 20μM, almost all organoids die. The effect is better than sunitinib, and it is expected to be used in the treatment of clinical TFE3 gene rearrangement renal cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1A graph of the inhibitory effects of different sertindole drug concentrations on TFE3-RCC cells was provided, and it was concluded that the drug concentration that inhibited 50% of the cells was 12.5 μM;

[0020] Figure 2 A graph of cell proliferation at different time points when the sertindole concentration was 13 μM compared with the control group was provided, and it was concluded that sertindole could significantly inhibit cell proliferation after 48 hours of application;

[0021] Figure 3 A graph showing the inhibitory effect of sertindole on cell proliferation at a concentration of 13 μM compared to the control group was provided, showing that sertindole can significantly inhibit cell proliferation and even completely kill tumor cells;

[0022] Figure 4 A graph is provided showing the inhibitory effect of sertindole on cell migration ability when the concentration of sertindole is 13 μM compared with the control group. The cell migration rate of the control group is about 53%, while the cell migration rate of the sertindole group is about 35%. It is concluded that sertindole can significantly inhibit the migration ability of cells;

[0023] Figure 5 A graph is provided showing the inhibitory effect of sertindole on cell migration and invasion when the concentration of sertindole is 13 μM compared with the control group, and it is concluded that sertindole can significantly inhibit the migration and invasion of cells;

[0024] Figure 6 A graph of the inhibitory effects of different sertindole drug concentrations on TFE3-RCC organoids was provided, showing that the drug concentration that inhibited 50% of cells was 0.93 μM, and the maximum inhibitory efficiency could reach 99.99%;

[0025] Figure 7 A graph of the inhibitory effect of different sunitinib concentrations on TFE3-RCC organoids was provided, showing that the drug concentration that inhibited 50% of cells was 0.06 μM, and the maximum inhibitory efficiency could reach 57.85%;

[0026] Figure 8 Brightfield images of the therapeutic effects of TFE3-RCC organoids treated with the control, sertindole, and sunitinib groups are provided. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0028] The purpose of the present invention is to explore a small molecule drug that can target and treat TFE3 gene rearrangement renal cell carcinoma. In order to achieve the above purpose, the inventor introduced a small molecule drug Sertindole (Chinese name: Sertindole) that can effectively inhibit the proliferation, migration and invasion of TFE3 gene rearrangement renal cell carcinoma. At the same time, the therapeutic effect of the drug and sunitinib in TFE3 gene rearrangement renal cell carcinoma was studied at the organoid level. It was found that the therapeutic effect of Sertindole (Chinese name: Sertindole) was significantly better than that of sunitinib.

[0029] Sertindole is an atypical antipsychotic drug. Its English name is Sertindole, its trade name is Serdolect (Serlect), and its chemical name is: 1-[2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl-2-imidazolidinone. Its English name is: 5-Chloro-1-(4-fluorophenyl)-3-[1- (2-oxoimidazolidin-1-ylethyl)-4-piperidyl]-1H-indole; or 1-[2-[4-[5-Chloro-1- (4-fluorophenyl)-1H-indol-3-y1]piperidinyl]ethyl]-2-imidazolidinone. This compound is disclosed in US 4,710,500, and its anti-schizophrenia activity is disclosed in US 5,112,838. In vivo, sertindole is an antagonist acting at D2 and 5-HT2A receptors in the central nervous system and has further demonstrated therapeutic efficacy in models of anxiety, hypertension, drug abuse, and cognitive impairment.

[0030] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1 Cell half inhibitory concentration (IC 50 ) Determination

[0032] TFE3 gene rearranged renal cell carcinoma cells with good normal culture status, among which the TFE3 gene rearranged renal cell carcinoma cell line was obtained by primary isolation and culture of renal carcinoma tissue and immortalization at the Urology Department of the Third Medical Center of the General Hospital of the People's Liberation Army. The specific steps are as follows: 1. Prepare collagenase digestion solution: Mix equal volumes of type I collagenase and type II collagenase with a concentration of 2 mg / ml to prepare collagenase digestion solution for digesting tissue cells. 2. Prepare primary cell culture medium: Mix 20% fetal bovine serum, 2% penicillin-streptomycin and DMEM / F12 culture medium to prepare primary cell culture medium. 3. Disintegrate tumor tissue: Take a soybean-sized tumor tissue in a 1.5 ml centrifuge tube, take 500 µl of collagenase digestion solution and add it to the centrifuge tube, and then use autoclaved tissue scissors to repeatedly cut the tissue into pieces until the diameter is about the size of a sesame seed. 4. Initial tissue cell digestion: Place the centrifuge tube containing tissue cells and collagenase digestion solution in a 37 °C incubator for 5 min, centrifuge at 3000 rpm for 3 min, discard the upper layer of collagenase digestion solution, and obtain the digested tissue cells. 5. Re-crushing tissue cells and digestion: Resuspend the tissue cells obtained in the above steps with 1 ml of collagenase tissue digestion solution, and use autoclaved tissue scissors to repeatedly chop the tissue until there is no obvious resistance when using a 1 ml pipette to absorb the liquid. Place the centrifuge tube in a 37 °C incubator for 10 min, centrifuge at 3000 rpm for 3 min, discard the upper layer of collagenase digestion solution, and resuspend the tissue cell group with the prepared culture medium. 6. Pass the suspended tissue cell suspension through a 100 µm sterile filter and inoculate it into a T25 culture flask for culture. 7. Immortalization of primary cells: Observe the cell growth status daily, and when the fusion rate of normally growing adherent cells reaches 50%, transfect the primary cells with SV40 large T antigen stable lentivirus (provided by Hanyin Biotechnology Co., Ltd.), and continue to passage for about 20 generations to establish a stable immortalized cell line. 8. Identification of TFE3 rearranged renal cell carcinoma immortalized cell line: Identify the expression of renal tissue marker PAX8 and TFE3 protein by immunohistochemistry, and identify it as a TFE3 rearranged renal cell carcinoma cell line by FISH, and identify the isolated primary immortalized cell line TFE301-1 cell line as NONO-TFE3 fusion type by genome sequencing technology.

[0033] After trypsin digestion, about 2000 cells / well were counted on a cell counting plate and plated into a 96-well plate. After 24 hours of cell attachment, different concentrations of the drug sertindole (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM) were added. After 48 hours of incubation at 37 degrees Celsius, cell activity detection reagent (CCK-8) was added and continued to be incubated. The absorbance value was measured after 2 hours.

[0034] Result analysis:

[0035] The experimental results can be found in Figure 1 . Figure 1 The activity of sertindole in TFE3 gene rearrangement renal cell carcinoma cells at concentrations of 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM is shown. The drug concentration of sertindole that kills half of the TFE3 gene rearrangement renal cell carcinoma cells, i.e., IC 50 is 12.5 μM.

[0036] Example 2 Cell proliferation assay

[0037] 1). Cell viability assay reagent (CCK-8): TFE3 gene rearrangement renal cell carcinoma cells in good normal culture state were digested with trypsin and counted on a cell counting plate at about 2000 cells / well. They were plated into 96-well plates and divided into two groups (control group DMSO and experimental group sertindole). DMSO and sertindole (13 μM) were added after the cells adhered to the plate for 24 hours. The cell absorbance values ​​were detected at 0 h, 24 h, and 48 h after the addition of the drugs, and the relative cell viability was calculated.

[0038] 2). Plate cloning experiment: TFE3 gene rearrangement renal cell carcinoma cells in good normal culture state were digested with trypsin and counted on a cell counting plate at about 2000 cells / well. They were plated into six-well plates and divided into two groups (control group DMSO and experimental group sertindole). DMSO and sertindole (13 μM) were added respectively 24 hours after plating. After 10 days, 4% tissue cell fixative was added and fixed for 10 minutes. The fixative was then discarded and crystal violet solution was added for staining to observe the cell colony formation.

[0039] Result analysis:

[0040] The experimental results can be found in Figure 2 and Figure 3 .from Figure 2 It can be seen that compared with the control group, the sertindole drug concentration of 13 μM can significantly inhibit the activity of TFE3 gene rearrangement renal cell carcinoma cells after 48 hours of treatment. Figure 3 It can be seen that compared with the control group, the treatment of TFE3 gene rearrangement renal cell carcinoma with a concentration of 13 μM of sertindole can significantly inhibit its proliferation ability.

[0041] Example 3 Cell migration and invasion assay

[0042] 1). Cell scratch test: TFE3 gene rearrangement renal cell carcinoma cells in good normal culture state were digested with trypsin and plated into six-well plates. They were divided into two groups (control group DMSO and experimental group sertindole). The drugs were treated for 48 hours respectively. After the cells were full, the scratch test was performed and the scratch width at 0 h was recorded. The specific steps of the scratch test were as follows: After the cells were plated, the cell growth density was observed. After the cells were completely full of the field of view, a straight line was drawn with a 200µl yellow gun tip with appropriate force. Then, PBS was washed off the cells that fell off the scratch and observed under a microscope. The coordinates and scratch width were recorded. After culturing in a 37°C incubator for 24 hours, the scratch width at the same coordinate was recorded. The formula was used: cell migration rate = (scratch width at 0 h - scratch width after 24 hours) / scratch width at 0 h × 100%.

[0043] 2). Cell migration: TFE3 gene rearrangement renal cell carcinoma cells in good normal culture state were digested with trypsin and resuspended in serum-free medium. About 30,000 cells were counted and evenly spread in the upper chamber. 10% serum medium was added to the lower chamber and cultured in an incubator at 37 degrees Celsius for 24 hours. The medium was then discarded and 4% tissue cell fixative was added. The cells were fixed for 10 minutes and then the fixative was discarded. Crystal violet solution was added for staining and the number of cells passing through the chamber was observed under a microscope and photographed to count.

[0044] The specific steps of adding sertindole and the control group are as follows: TFE3-RCC cells were plated in culture dishes, one group was added with sertindole at a concentration of 13 μM, and the other group was set as the control group. After 48 hours of culture, the cells were digested with trypsin and resuspended in serum-free medium. About 30,000 cells were counted and evenly plated in the upper chamber. 10% serum medium was added to the lower chamber and cultured in an incubator at 37 degrees Celsius for 24 hours. The medium was then discarded, 4% tissue cell fixative was added, fixed for 10 minutes, the fixative was discarded, crystal violet solution was added for staining, and the number of cells passing through the chamber was observed under a microscope and photographed.

[0045] 3). Invasion assay: Before the start of the experiment, matrigel was spread on the upper layer of the chamber (matrigel: serum-free medium = 1:8), and the chamber was placed in a 37°C incubator for 30 minutes before the same steps as the cell migration assay were performed.

[0046] Result analysis:

[0047] The experimental results can be found in Figure 4 and Figure 5 .from Figure 4 It can be seen that compared with the control group, the sertindole drug concentration of 13 μM in the treatment of TFE3 gene rearrangement renal cell carcinoma can significantly inhibit its migration ability. Figure 5It can be seen that compared with the control group, the treatment of TFE3 gene rearrangement renal cell carcinoma with a concentration of 13 μM sertindole can significantly inhibit its migration and invasion ability.

[0048] Example 4 Organoid Experiment

[0049] The half inhibitory concentration (IC 50 ) Assay: Organoids in good normal culture state (Beijing Daxiang Technology Co., Ltd.) were trypsin digested and centrifuged at 1000 rpm at room temperature. The cell pellets were resuspended in culture medium and mixed evenly with 60% matrix gel, and inoculated into the microwells of IBAC S1 chip (Beijing Daxiang Technology Co., Ltd.) at a standard of 800 cells / 5μL / well. Organoids were cultured in organoid culture medium for 3 days, and then incubated in organoid culture medium with drugs for 5 days (the concentration gradients of sertindole drugs were: 0.37 μM, 1.11 μM, 3.33 μM, 10 μM, 30 μM, respectively; the concentration gradients of sunitinib drugs were: 0.002 μM, 0.007 μM, 0.02 μM, 0.06 μM, 0.184 μM, respectively). Then, the bright field state of the organoids was recorded using an inverted microscope and photographed for preservation.

[0050] After the drug incubation of organoids is completed, the ATP activity detection method is used. The old culture medium is discarded, and the organoid activity detection reagent is added and incubated at room temperature in the dark for 30 minutes. Then, the chemiluminescence value is detected by a multifunctional microplate reader to evaluate the activity of the organoids.

[0051] Result analysis:

[0052] The experimental results can be found in Figures 6 to 8 .from Figure 6 It can be seen that the activity of TFE3 gene rearranged renal cell carcinoma organoids corresponding to sertindole at concentrations of 0.37 μM, 1.11 μM, 3.33 μM, 10 μM, and 30 μM. It can be seen that the drug concentration of sertindole that kills half of the TFE3 gene rearranged renal cell carcinoma organoids is IC 50 The maximum inhibition rate was 0.93 μM and 99.99%.

[0053] from Figure 7 It can be seen that the activity of TFE3 gene rearranged renal cell carcinoma organoids corresponding to sunitinib at concentrations of 0.002 μM, 0.007 μM, 0.02 μM, 0.06 μM, and 0.184 μM. It can be seen that the drug concentration at which sertindole kills half of the TFE3 gene rearranged renal cell carcinoma organoids is IC 50 The maximum inhibition rate was 0.05 μM and 57.85%.

[0054] from Figure 8It can be seen that compared with the control group, sertindole can significantly inhibit the growth of TFE3 gene rearrangement renal cell carcinoma organoids, and sunitinib can inhibit the growth of TFE3 gene rearrangement renal cell carcinoma organoids to a certain extent, but compared with sertindole, its inhibitory effect is significantly weaker.

[0055] Although several embodiments of the present invention have been given herein, those skilled in the art should understand that the embodiments of the present invention may be modified without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used as a limitation on the scope of the present invention.

Claims

1. Use of sertindole in the preparation of a drug for treating TFE3 gene rearrangement renal cell carcinoma.

2. The use according to claim 1, characterized in that The medicament further includes a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 2, characterized in that: The carrier or excipient includes one or more of a binder, a filler, a diluent, a tabletting agent, a lubricant, a disintegrant, a colorant, and a wetting agent.

4. The use according to claim 1, characterized in that The dosage form of the drug is tablet, capsule, oral liquid, lozenge, granule, pill, powder, granule, ointment, suspension, powder, solution, injection, cream, spray, drop or patch.

5. The use according to claim 1, characterized in that: The administration route of the drug is oral, transdermal, intramuscular, subcutaneous or intravenous injection.

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