Application of padsevonil in the preparation of drugs for treating TFE3 rearranged renal cell carcinoma

Abstract

The present invention provides the use of padsevonil in the preparation of a drug for treating TFE3 rearrangement renal cell carcinoma, and relates to the field of biomedicine technology. The use of padsevonil in the preparation of a drug for treating TFE3 rearrangement renal cell carcinoma. The present invention provides a new drug padsevonil for treating TFE3-RCC, so as to delay the malignant progression of TFE3-RCC and improve the survival of patients. The drug padsevonil provided by the present invention can effectively inhibit the proliferation, invasion and migration of TFE3 rearrangement renal cell carcinoma cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of padsevonil in preparing a medicine for treating TFE3 rearrangement renal cell carcinoma. Background Art

[0002] TFE3-rearranged renal cell carcinoma (TFE3-RCC) is a rare type of renal cancer with a high degree of malignancy, accounting for about 40% of pediatric renal cell carcinoma cases and 5% of adult renal cell carcinoma cases. TFE3-RCC is a malignant renal lesion caused by the translocation of the short arm of the X chromosome (Xp11), which causes the TFE3 gene located on the short arm of the X chromosome to fuse with its partner gene. TFE3-RCC is highly invasive and malignant, and has a tendency to metastasize when the primary tumor is very small. Currently, there is no effective treatment for patients with advanced translocation renal cancer, and patients with distant metastasis and young patients usually have a poor prognosis.

[0003] Patients with recurrent and advanced TFE3-RCC often have a poor prognosis due to the lack of effective therapeutic targets. Many studies have shown that TFE3-RCC is relatively insensitive to targeted therapy or immunotherapy. Therefore, further searching for safe and effective new drugs to treat TFE3-RCC has become a scientific problem that needs to be solved urgently in the field of TFE3-RCC. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides an application of padsevonil in the preparation of a drug for treating TFE3 rearranged renal cell carcinoma. The technical solution is as follows:

[0005] Use of padsevonil in preparing a drug for treating TFE3 rearrangement renal cell carcinoma.

[0006] A drug or a drug composition for treating TFE3 rearranged renal cell carcinoma, comprising padsevonil.

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

[0012] The present invention provides a new drug padsevonil for treating TFE3-RCC, so as to delay the malignant progression of TFE3-RCC and improve the survival of patients. The drug padsevonil provided by the present invention can effectively inhibit the proliferation, migration and invasion of TFE3 rearranged renal cell carcinoma cells. CCK8 experiment shows that after adding padsevonil for 72 hours, the absorbance of DMSO group: padsevonil group = 2.24: 1.0; the scratch experiment shows that after adding padsevonil, the migration distance of TFE301-1 cells within 24 hours is DMSO group: padsevonil group = 538 μm: 282.0 μm; the transwell experiment shows that the number of TFE301-1 cells penetrating the transwell chamber membrane within 24 hours is DMSO group: padsevonil group = 1055.2: 105.0, and the number of TFE301-1 cells penetrating the transwell chamber membrane and matrix gel within 24 hours is DMSO group: padsevonil group = 1055.8: 96.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] Figure 1A is a bright field image provided by Example 1 of the present invention; Figure 1B This is a diagram of drug sensitivity test results provided in Example 2 of the present invention;

[0015] Figure 2A This is a graph of CCK8 experimental results provided in Example 3 of the present invention; Figure 2B is a scratch test result diagram provided in Example 4 of the present invention; Figure 2C This is a diagram of the Transwell experiment results provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0016] 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.

[0017] Since patients with recurrent and advanced TFE3-RCC are relatively insensitive to targeted therapy or immunotherapy and lack effective treatment approaches, the prognosis of patients is often poor. The present invention aims to provide a new drug padsevonil for treating TFE3-RCC to delay the malignant progression of TFE3-RCC and improve the survival of patients.

[0018] The molecular formula of Padsevonil is C 14 H 14 CIF 5 N 4 O 2 S, English name is (R)-4-(2-chloro-2,2-difluoroethyl)-1-((2-(methoxymethyl)-6-(trifluoromethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyl)pyrrolidin-2-one, molecular weight is 432.80, CAS number is 1294000-61-5.

[0019] The research data of the present invention show that targeting SV2B can inhibit the malignant progression of TFE3-RCC. Padsevonil can bind to SV2B with high affinity. A phase III clinical trial showed that humans have good tolerance and safety for padsevonil, suggesting that padsevonil has important clinical application prospects in the treatment of TFE3-RCC.

[0020] Embodiment 1, bright field photography.

[0021] 1) Approximately 100,000 TFE301-1 cells (source can be found in the article Feng H, Cao S, Fu S, Liu J,Gao Y, Dong Z, et al. NMRK2 is an efficient diagnostic indicator for Xp11.2 translocation renal cell carcinoma. J Pathol 2024; 264(2):228-240.) were plated into a 6-well plate.

[0022] 2) After 24 hours, change the medium and record the density and status of the cells under the bright field microscope at 0 hour.

[0023] 3) Add DMEM / F12 medium containing 2% serum and add padsevonil (HY-109009, MCE) according to the following drug concentration gradient: 0, 4, 8, 16, 32, 64 μM. Incubate for 24 hours.

[0024] 4) Record the density and status of cells under a bright field microscope for 24 hours.

[0025] Result analysis: The results are as follows Figure 1A As shown, with the increase of padsevonil drug concentration, the inhibitory effect on TFE301-1 cells became more obvious. Treatment with 32 μM padsevonil for 24 hours could significantly inhibit the proliferation of TFE301-1 cells.

[0026] Example 2, IC50 measurement

[0027] 1) Plate 10,000 TFE301-1 cells into a 96-well plate.

[0028] 2) After 24 hours, the medium was changed and DMEM / F12 medium containing 2% serum was added, and padsevonil was added according to the following drug concentration gradient: 0, 4, 8, 16, 32, 64 μM. Each concentration was repeated for 5 wells and cultured for 24 hours.

[0029] 3) Add 20 μL of CCK reagent (CK001-3000T, Langbolide) to each well, incubate at 37°C for 2 h, measure the absorbance at a wavelength of 450 nm, record the OD value, and remove the maximum and minimum values ​​in the 5 replicate wells.

[0030] 4) Draw a drug sensitivity IC50 curve.

[0031] The experimental results are as follows Figure 1B As shown, with the increase of padsevonil drug concentration, the inhibitory effect on TFE301-1 cells became more obvious. After calculation, the IC50 of padsevonil on TFE301-1 cells was about 40 μM.

[0032] Example 3, CCK8 experiment

[0033] 1) TFE301-1 cell plating (96-well plate): Divide into DMSO group and padsevonil group, with 5 replicate wells in each group, padsevonil concentration is 20 μM, and each well has approximately 5,000 cells. Each well has 100 μL of culture medium.

[0034] 2) Measure the OD values ​​corresponding to the four time points of 0, 24, 48, and 72 h. Add 20 μL of CCK8 reagent (CK001-3000T, Langbolide) before each measurement, incubate at 37°C for 2 h, measure the absorbance at a wavelength of 450 nm, record the OD value, and remove the maximum and minimum values ​​in the five replicate wells.

[0035] 3) Data analysis.

[0036] The experimental results are as follows Figure 2A As shown, from Figure 2A It can be seen that after adding padsevonil, the proliferation rate of TFE301-1 cells can be significantly inhibited. After adding padsevonil for 72 hours, the absorbance ratio of padsevonil group: DMSO group was 1.0±0.11:2.24±0.11, and the P value was less than 0.001, which was statistically significant. The above shows that padsevonil can inhibit the proliferation of TFE301-1 cells.

[0037] Example 4. Scratch test

[0038] 1) TFE301-1 cells were plated (6-well plate) so that the density of cells attached to the plate was approximately 90% after 24 hours.

[0039] 2) Scratching: Use the tip of a yellow gun to evenly scratch a “well” shape in the culture dish.

[0040] 3) Take photos to record the width of the scratch at 0 hours. At the same time, the holes after scratching were divided into two groups, namely DMSO group and padsevonil group, where the drug concentration of padsevonil group was 20 μM.

[0041] 4) After 24 hours, the width of the scratch remaining after TFE301-1 cells migrated for 24 hours was photographed.

[0042] 5) Analysis and statistics.

[0043] The experimental results are as follows Figure 2B As shown, from Figure 2B It can be seen that the addition of padsevonil can significantly inhibit the migration of TFE301-1 cells. Within 24 hours, the migration distance of TFE301-1 cells decreased from 538±9.7 μm (DMSO group) to 282.0±8.0 μm (padsevonil group), and the P value was less than 0.01, which was statistically significant. The above shows that padsevonil can inhibit the migration of TFE301-1 cells.

[0044] Example 5. Transwell experiment

[0045] 1) Matrigel plating. The migration assay does not require plating, but the invasion assay does. Take out the Matrigel (354234, Corning) from -80℃ 12 h in advance and place it in a 4℃ refrigerator for later use. Add 50 μL of Matrigel mixture (Matrigel: Culture medium = 6:4, Matrigel from Corning, Catalog No. 354234, Culture medium from Procell, Catalog No. PM150210) to the chamber where the plating is required, and place it in a cell culture incubator for 60 min. Do not generate bubbles during the plating process.

[0046] 3) Add 600 μL of DMEM / F12 medium containing 10% serum to the bottom of the chamber; digest the TFE301-1 cells and prepare a single-cell suspension (without serum).

[0047] 4) Add 200 μL of cell suspension to each chamber, count the cells to ensure that the number of cells in 200 μL of suspension is about 50,000 (without gel) or 70,000 (with gel), and place in a cell culture incubator for culture.

[0048] 5) Proceed to the next step after 24 hours.

[0049] 6) Aspirate the culture medium inside and below the chamber, and gently soak the lower chamber with PBS to remove the culture medium and serum inside and outside the chamber as much as possible; soak the chamber in tissue cell fixative (P11102, Solebow) at room temperature for 20 min.

[0050] 7) Soak the chamber in PBS twice to remove the fixative inside and outside the chamber; soak the chamber in crystal violet working solution for 5 min, and soak it in running tap water to remove the crystal violet.

[0051] 8) Use a cotton swab to clean the adhered and fixed cells in the chamber, take pictures under a microscope, and count them.

[0052] The experimental results are as follows Figure 2C As shown, from Figure 2C It can be seen that within 24 hours, the number of TFE301-1 cells that penetrated the transwell membrane decreased from 1055.2±105.2 (DMSO group) to 105.0±24.5 (padsevonil group), with a P value less than 0.001, which was statistically significant; within 24 hours, the number of TFE301-1 cells that penetrated the transwell membrane and matrix gel decreased from 1055.8±182.5 (DMSO group) to 96.6±18.6 (padsevonil group), with a P value less than 0.001, which was statistically significant. The above shows that padsevonil can inhibit the migration and invasion of TFE301-1 cells.

[0053] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of padsevonil in the preparation of a drug for treating TFE3 rearranged renal cell carcinoma.

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