Use of CZC-54252 in the preparation of a drug for treating esophageal squamous cell carcinoma

Abstract

The application belongs to the field of medicine, and particularly relates to application of CZC-54252 in preparation of a drug for treating esophageal squamous cell carcinoma. The results of the application show that CZC-54252 can efficiently inhibit expression of RSK4 enzyme, and can efficiently inhibit proliferation, invasion and migration of esophageal squamous cell carcinoma cells. In combination with Afatinib, the proliferation of an esophageal squamous cell carcinoma erlotinib-resistant strain can be significantly inhibited, and the results provide an effective technical means for treatment and prognosis of esophageal squamous cell carcinoma patients.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of CZC-54252 in the preparation of drugs for treating esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer. Despite the availability of various treatment options for ESCC, the 5-year overall survival rate for advanced ESCC is only 15%. Radiation therapy is an important treatment for ESCC, especially for patients with surgically unresectable advanced esophageal cancer. Unfortunately, radioresistance to tumor cells leads to recurrence and treatment failure in ESCC; most patients experience recurrence even after pathological remission, and radiation therapy also causes various side effects. Due to the limitations of radiation therapy, the development of targeted therapies for ESCC is essential.

[0003] The main targets of molecular targeted therapy for ESCC include epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (VEGF). However, research on targeted therapy is still in its early stages, and the precise therapeutic targets for ESCC are not yet fully understood. Furthermore, the emergence of drug resistance renders normal doses of targeted drugs ineffective in suppressing cancer. Therefore, the development of new chemotherapy drugs is of great significance for the treatment and prognosis of ESCC patients.

[0004] CZC-54252 is an effective selective LRRK2 inhibitor, but there are currently no reports of its use in esophageal squamous cell carcinoma. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for CZC-54252.

[0006] In a first aspect, the present invention provides the use of CZC-54252 in the preparation of a medicament for treating esophageal squamous cell carcinoma.

[0007] Furthermore, the CZC-54252 inhibits the proliferation of esophageal squamous cell carcinoma cells.

[0008] Furthermore, the CZC-54252 inhibits the invasion and migration of esophageal squamous cell carcinoma cells.

[0009] Furthermore, CZC-54252 reduces drug resistance in esophageal squamous cell carcinoma cells.

[0010] Furthermore, the CZC-54252, in combination with Afatinib, is used to prepare a drug for treating esophageal squamous cell carcinoma.

[0011] In a second aspect, the present invention provides a medicament for treating esophageal squamous cell carcinoma, the medicament comprising an effective dose of CZC-54252.

[0012] Furthermore, CZC-54252 is the sole active ingredient or one of the active ingredients of the drug.

[0013] Furthermore, the drug inhibits the proliferation, invasion, and migration of esophageal squamous cell carcinoma cells.

[0014] Thirdly, the present invention provides a drug combination for treating esophageal squamous cell carcinoma, the drugs comprising an effective dose of CZC-54252 and Afatinib.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention provides a new use for CZC-54252. The research results of this invention show that CZC-54252 can effectively inhibit the expression of RSK4 enzyme and can effectively inhibit the proliferation, invasion and migration of esophageal squamous cell carcinoma cells. When used in combination with Afatinib, it can significantly inhibit the proliferation of erlotinib-resistant strains of esophageal squamous cell carcinoma. This result provides an effective technical means for the treatment and prognosis of ESCC patients. Attached Figure Description

[0017] Figure 1 The chemical structural formula of CZC-54252 is given.

[0018] Figure 2 The curve shows the half-inhibition curve of CZC-54252 on RSK4 enzyme.

[0019] Figure 3 The half-inhibition curve of CZC-54252 against esophageal squamous cell carcinoma cells (TE-10) is shown.

[0020] Figure 4 The growth curve of esophageal squamous cell carcinoma cells (TE-10) treated with 3μm CZC-54252.

[0021] Figure 5 The effect of CZC-54252 on the invasion and migration of esophageal squamous cell carcinoma cells (TE-10) is shown in Figure A, where A is the Giemsa staining pattern of cells and B is the quantitative statistical result of A.

[0022] Figure 6The effects of different treatments on the proliferation of erlotinib-resistant esophageal squamous cell carcinoma strain KYSE450-IR were investigated. Among them, a was DMSO treatment, b was 2 μm Afatinib treatment, c was 2 μm CZC-54252 treatment, and d was Afatinib + CZC-54252 treatment. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] This invention discloses a new use for CZC-54252, wherein the molecular formula of CZC-54252 is C 22 H 25 ClN6O4S, CAS No.: 1191911-27-9, chemical structural formula as follows: Figure 1 As shown.

[0025] Example 1: Inhibition of RSK4 enzyme by CZC-54252

[0026] 1 Experimental Methods

[0027] In this experiment, the ADP-Glo ​​method was used to detect the effect of CZC-54252 on RSK4 enzyme. The initial concentration of CZC-54252 was 10 μM, with 5-fold serial dilutions, 2 replicates, and 6 concentrations.

[0028] (1) Thaw RSK4 enzyme, RSK Substrate, kinase assay buffer III (5× buffer), DTT (0.1M) and ATP (10mM) on ice, and keep all of the above reagents on ice throughout the experiment.

[0029] (2) Prepare a 1× buffer solution by mixing 5× buffer solution with deionized water and add DTT to it. The concentration of DTT in the 1× buffer solution is 50 μM.

[0030] (3) Add 1 μl / well of the compound to be tested to a white microplate and centrifuge the microplate at 1000 rpm for 1 minute.

[0031] Positive control wells (Pos.Ctrl): 1 μl / well of compound dilution solvent;

[0032] Blank control wells: 1 μl / well 1× buffer.

[0033] (4) After the RSK4 enzyme is completely thawed, dilute the RSK4 enzyme to 1 ng / μl with 1× buffer, and add 2 μl / well to a white microplate. At this time, the amount of RSK4 enzyme in each well is 2 ng. Add 2 μl / well of 1× buffer to the blank control well. This step should be performed on ice. After adding, centrifuge the microplate at 1000 rpm for 1 minute.

[0034] (5) Prepare RSK Substrate / ATP mixture:

[0035] RSK Substrate / ATP Mixture: Add 130 μl of RSK Substrate (1 mg / ml) to 3.25 μl of 5 mM ATP and 127 μl of 2× buffer (note that this is a proportional dilution). At this point, the ATP concentration is 62.5 μM and the RSK Substrate concentration is 0.5 mg / ml. This step should be performed on ice.

[0036] (6) Take 2 μl / well of the RSK Substrate / ATP mixed solution into a white microplate. At this time, the concentration of RSK Substrate is 0.2 mg / ml and the concentration of ATP is 25 μM. After adding the solution, centrifuge the microplate at 1000 rpm for 1 minute.

[0037] (7) After centrifugation, attach the membrane to the microplate, press the membrane firmly, and incubate at 25°C for 1 hour.

[0038] (8) Equilibrate the ADP-Glo™ reagent and Kinase Detection-related reagents required in the Promega kit to room temperature, and mix the Kinase Detection buffer and Kinase Detection Substrate according to the instructions for later use.

[0039] (9) After the incubation is complete, take 5 μl / well of ADP-Glo™ reagent and add it to a white microplate. Centrifuge the microplate at 1000 rpm for 1 minute and incubate at 25°C for 40 minutes.

[0040] (10) After the incubation is over, take 10 μl of Kinase Detection mixture per well and add it to the microplate. Centrifuge the microplate at 1000 rpm for 1 minute and incubate at 25°C for 30 minutes.

[0041] (11) After incubation, perform chemiluminescence detection on a plate reader and read the luminescence value (RLU);

[0042] (12) Calculation of enzyme inhibition rate:

[0043] %Inhibition=100-(RLU(Sample)-RLU(Blank)) / (RLU(Pos.Ctrl)-RLU(Blank))×100%.

[0044] 2 Experimental Results

[0045] like Figure 2 As shown, the inhibitory effect of CZC-54252 on RSK4 kinase activity increased with increasing CZC-54252 concentration. Curve fitting using GraphPad software yielded an IC50 value of 63.39 nM. These results indicate that CZC-54252 effectively inhibits RSK4 phosphorylation and activation, making it a potent RSK4 inhibitor.

[0046] Example 2: Regulation of esophageal squamous cell carcinoma cell growth by CZC-54252

[0047] 1 Experimental Methods

[0048] In this experiment, esophageal squamous cell carcinoma cell line (TE10) was stimulated with different concentrations of CZC-54252. The effect of CZC-54252 on the proliferation of esophageal squamous cell carcinoma cells was detected by CCK8 assay, and the effect of CZC-54252 on the migration of esophageal squamous cell carcinoma cells was detected by Transwell assay. At the same time, no drug was used as a blank control (Black or Vector).

[0049] 1.1 CCK8 testing steps:

[0050] The procedure was performed using the CCK-8 cell proliferation kit from Shanghai Taoshu Biotechnology Co., Ltd.

[0051] (1) 96-well plates were seeded with cell suspension, 100 μL per well, 2,000 cells per well.

[0052] (2) Cultivate or administer drugs as required by the experiment and treat for an appropriate period of time.

[0053] (3) Add 10 μL of CCK-8 solution to each well and incubate at 37°C.

[0054] (4) Use an ELISA reader with a wavelength of 450 nm to measure the absorbance. Plot a growth curve using the time and absorbance values.

[0055] 1.2 Cell migration:

[0056] (1) Digest the cultured TE10 cells to prepare a single-cell suspension and wash them with PBS.

[0057] (2) The control group was resuspended in DMEM medium containing DMSO to a final volume of 5 × 10⁻⁶. 5The experimental group was resuspended in 3 μM CZC-54252 DMEM medium to a concentration of 5 × 10⁻⁶ cells / ml. 5 per ml.

[0058] (3) Add 500 μl of DMEM medium containing 10% fetal bovine serum to the lower chamber of the 24-well plate, and add 200 μl of cell resuspension of the experimental group and the control group to the upper chamber respectively.

[0059] (4) Routine culture for 24 hours.

[0060] (5) Remove the culture medium from the chamber, fix the cells in the chamber with anhydrous methanol for 15 min, and rinse with PBS.

[0061] (6) Add Giemsa staining solution to the 24-well plate, stain for 20 min, and wash with PBS.

[0062] (7) Carefully wipe away the inner layer of cells with a cotton swab and air dry the membrane.

[0063] (8) Take pictures under a microscope, count 5 high-power fields, and take the average value.

[0064] 1.3 Cell invasion:

[0065] (1) Place the BD Matrigel gel, which is frozen in a -80℃ freezer, at 4℃ overnight to thaw it into a liquid state.

[0066] (2) Dilute Matrigel to 50 μg / ml using serum-free culture medium at a concentration of 1:8. After thorough mixing, add 60 μl of Matrigel gel to the upper chamber of the Transwell chamber.

[0067] (3) Digest the cultured TE10 cells to prepare a single-cell suspension and wash them with PBS.

[0068] (4) The control group was resuspended in DMEM medium supplemented with DMSO to a final volume of 5 × 10⁻⁶. 5 The experimental group was resuspended in 3 μM CZC-54252 DMEM medium to a concentration of 5 × 10⁻⁶ cells / ml. 5 per ml.

[0069] (5) Add 500 μl of DMEM medium containing 10% fetal bovine serum to the lower chamber of the Transwell chamber, and add 200 μl of cell resuspension of the experimental group and the control group to the upper chamber respectively.

[0070] (6) Place the Transwell chamber in a constant temperature cell culture incubator and culture for 24 hours.

[0071] (7) After the culture is completed, remove the Transwell chamber and wipe the upper chamber with a cotton swab dipped in PBS to remove the Matrigel gel.

[0072] (8) Place the chamber in methanol and fix for 20 min;

[0073] (9) Add Giemsa staining solution to 24-well plate, stain for 20 min, and wash with PBS.

[0074] (10) Take pictures under a microscope, count 5 high-power fields, and take the average value.

[0075] 2 Experimental Results

[0076] like Figure 3-5 As shown, the half-maximal inhibitory concentration (WMC) of CZC-54252 against TE10 cells was 2.494 μM. Compared with the blank control, CZC-54252 significantly inhibited the proliferation, invasion, and migration of TE10 cells.

[0077] Example 3: Regulation of proliferation of esophageal squamous cell carcinoma resistant to icotinib (KYSE450-IR) by CZC-54252 combined with afatinib

[0078] 1 Experimental Methods

[0079] The expression levels of EGFR and their sensitivity to icotinib in various ESCC cell lines (T1, T10, T11, KYSE150, KYSE450, ECA109, EC9706) were investigated. The KYSE450 cell line, which highly expresses EGFR and is sensitive to icotinib (low IC50), was selected. Icotinib resistance was induced in KYSE450 cells using an escalating icotinib concentration regimen, resulting in a stable icotinib-resistant cell line. Based on literature reports and preliminary experiments conducted before formal induction culture, the optimal icotinib concentration gradient for induction was determined to be 100 nM, 300 nM, 500 nM, 800 nM, 1 μM, and 5 μM. The initial induction dose in this experiment was 100 nM, and the maximum induction dose was 5 μM. The icotinib-containing culture medium was changed every two days during induction. After two weeks of induction at the initial induction dose, and once cell growth stabilized, the drug induction dose was gradually increased, with each dose maintained for 14 days until the maximum induction dose was reached. The drug-resistant cells obtained after 10 months of induction culture were named KYSE450-IR.

[0080] KYSE450-IR cells were stimulated with CZC-54252 (2μm), Afatinib (2μm), or CZC-54252+Afatinib (2μm, with a mass ratio of 1:1), with DMSO as a blank control. The effects of different drugs on KYSE450-IR cell proliferation were detected by CCK8 assay (method as in Example 2).

[0081] 2 Experimental Results

[0082] like Figure 6 As shown, both CZC-54252 and Afatinib alone can inhibit the proliferation of KYSE450-IR, but the inhibitory effect of Afatinib is extremely low, indicating that KYSE450-IR has developed resistance to Afatinib. However, when CZC-54252 and Afatinib are used in combination, the inhibitory effect on KYSE450-IR is significantly stronger than that of CZC-54252 alone, indicating that CZC-54252 and Afatinib can play a synergistic role. This result provides a new approach for the treatment of esophageal squamous cell carcinoma.

[0083] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0084] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of CZC-54252 in combination with Afatinib in the preparation of a drug for treating esophageal squamous carcinoma resistant to Icotinib, characterized in that, The structural formula of CZC-54252 is shown below: 。

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