CN118615268A - Application of fingolimod in the preparation of cancer therapeutic drugs - Google Patents

Abstract

The present invention belongs to the field of medicine, and specifically relates to the use of fingolimod in the preparation of cancer therapeutic drugs. The cancer includes esophageal squamous cell carcinoma. The present invention finds that fingolimod can significantly reduce the vitality of esophageal squamous cell carcinoma cells, and has the potential to be developed into a therapeutic drug for esophageal squamous cell carcinoma; the present invention also finds that fingolimod can increase the radiotherapy sensitivity and chemotherapy sensitivity of esophageal squamous cell carcinoma resistant cell lines, indicating that fingolimod can be used as a good solution to solve the difficult tumor resistance and radiotherapy resistance problems in the field of esophageal squamous cell carcinoma treatment. The present invention will provide new treatment options for patients with esophageal squamous cell carcinoma and has broad application prospects.

Description

Application of fingolimod in the preparation of cancer therapeutic drugs

Technical Field

The present invention belongs to the field of medicine, and specifically relates to the application of fingolimod in the preparation of drugs for treating cancer.

Background Art

Esophageal cancer (EC) is one of the most common malignant tumors of the digestive tract, with 813,000 new cases of esophageal cancer worldwide in 2023. Esophageal cancer is divided into esophageal adenocarcinoma (EADC) and esophageal squamous cell carcinoma (ESCC). Surgery is the first choice for the treatment of ESCC, assisted by other treatments. For some patients who cannot undergo surgery, radiotherapy becomes the first choice for ESCC patients, but many patients are insensitive to radiotherapy or even develop radiotherapy resistance, which is a clinical problem that plagues doctors and patients. In addition, ESCC patients have severe resistance to platinum drugs, which is also one of the main reasons for poor prognosis.

Drugs that can increase the sensitivity of radiotherapy and chemotherapy are playing an increasingly important role in patients with clinical resistance to radiotherapy and chemotherapy. Fingolimod is a drug for the treatment of multiple sclerosis. It has a significant inhibitory effect on the proliferation of liver cancer, breast cancer, glioma, and colon cancer cells, but its role in fighting esophageal squamous cell carcinoma and reducing clinical esophageal squamous cell carcinoma radiotherapy resistance and chemotherapy resistance has not been reported.

Summary of the invention

The present inventors found in the process of studying tumor treatment that fingolimod has a good inhibitory effect on human esophageal squamous cell carcinoma cells KYSE450 and ECA109, with IC ₅₀ values of 50.23 nM and 72.28 nM, respectively (see Example 1).

During the research, the inventors conducted a study on the effect of fingolimod on improving the radiotherapy sensitivity and chemotherapy sensitivity of esophageal squamous cell carcinoma resistant cell lines. The results showed that the clone number of esophageal squamous cell carcinoma resistant cell lines treated with fingolimod combined with X-ray irradiation was significantly reduced compared with X-ray irradiation alone (see Example 3), indicating that fingolimod can increase the sensitivity of esophageal squamous cell carcinoma resistant cell lines to radiation. The IC ₅₀ value of esophageal squamous cell carcinoma resistant cell lines treated with fingolimod combined with cisplatin was significantly reduced compared with fingolimod combined with DMSO (see Example 4), indicating that fingolimod can increase the sensitivity of esophageal squamous cell carcinoma resistant cell lines to chemotherapy. In the DARTS experiment, fingolimod can bind to 7 specific peptide segments of the NUP54 protein (Example 5), indicating that the target of fingolimod against esophageal squamous cell carcinoma is the NUP54 protein.

Therefore, the first object of the present invention is to provide the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs, wherein the cancer includes esophageal squamous cell carcinoma.

Fingolimod (FTY720 free base) is a sphingosine1-phosphate (S1P) antagonist that acts on K562 and NK cells with an IC ₅₀ of 0.033nM. Fingolimod is also a pak1 activator and immunosuppressant, CAS No.: 162359-55-9.

According to one aspect of the present invention, the drug is used to promote cancer cell apoptosis, reduce cancer cell activity, inhibit cancer cell proliferation, and suppress cancer cell migration.

According to one aspect of the present invention, the application is the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of a drug for improving the chemotherapy sensitivity of edible resistant tubular squamous cell carcinoma.

According to one aspect of the present invention, the chemotherapy drug includes cisplatin. Cisplatin (DDP) is a Western medicine name. The commonly used preparation is an injection, which is an anti-tumor drug. It is used to treat small cell and non-small cell lung cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, melanoma, sarcoma, head and neck tumors and various squamous cell carcinomas and malignant lymphomas.

According to one aspect of the present invention, the application is the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of a drug for improving the radiotherapy sensitivity of drug-resistant esophageal squamous cell carcinoma cells.

Radiotherapy is the treatment of malignant tumors and some benign diseases using one or more ionizing radiations. The means of radiotherapy is ionizing radiation. The most commonly used direct ionizing particles in radiotherapy are electrons, and the most commonly used indirect ionizing particles are photons. The energy range of X-rays used in radiotherapy is 1-25MV.

According to one aspect of the present invention, the application is the application of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in combination with cisplatin in the preparation of a drug for treating esophageal squamous cell carcinoma. The fingolimod, its derivatives or pharmaceutically acceptable salts thereof and cisplatin are administered simultaneously or continuously in any order. When administered at the same time, the components in the composition are used in the same or independent formulations. When used at different times, the components of the composition can use the same or different dosage forms.

According to one aspect of the present invention, the terms "combination" or "combination use" or "co-administration" or "combination drug" used throughout the specification refer to the use of therapeutic agents included in the same or separate pharmaceutical preparations at the same time or different times.

Another object of the present invention is to provide a cancer treatment drug, wherein the cancer includes esophageal squamous cell carcinoma, and the active ingredients of the drug include cisplatin and fingolimod, its derivatives or pharmaceutically acceptable salts thereof. The drug also includes pharmaceutically acceptable excipients. The excipients include conventional diluents (such as water for injection, microcrystalline cellulose, etc. at least one), fillers (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, vaseline, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc. at least one), binders (such as carbomer, gum arabic, starch, cellulose, gelatin, polyvinyl pyrrolidone, polyacrylamide, etc. at least one), disintegrants (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc. at least one), lubricants (such as talc, hard At least one of magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micro-powdered silica gel, etc.), a wetting agent (such as at least one of propylene glycol, glycerol, ethanol, etc.), a stabilizer (such as at least one of disodium ethylenediaminetetraacetic acid, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, niacinamide, vitamin C, etc.), an osmotic pressure regulator (such as at least one of sodium chloride, glucose, etc.), a pH regulator (such as at least one of triethanolamine, sodium hydroxide, sodium citrate, etc.), a preservative (such as at least one of chlorobutanol, parabens, ethylparaben, benzalkonium bromide, etc.).

According to one aspect of the present invention, each drug in the anticancer preparation can be in the form of tablets, capsules, suspensions, solutions, injections, etc., either alone or together.

The present invention has the following beneficial effects:

(1) The present invention is the first to discover that fingolimod can be used to treat esophageal squamous cell carcinoma, providing a new use of fingolimod.

(2) The present invention discovered for the first time that fingolimod can increase the radiosensitivity and chemotherapy sensitivity of esophageal squamous cell carcinoma resistant cell lines, indicating that fingolimod can be used as a good solution to solve the difficult problems of tumor resistance and radioresistance in the treatment of esophageal squamous cell carcinoma.

(3) The present invention discovered through the DARTS experiment that the target of fingolimod in treating esophageal squamous cell carcinoma is NUP54. This result provides a theoretical basis for the targeted development of therapeutic drugs for esophageal squamous cell carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the half-inhibition curves of fingolimod on KYSE450 cells (A) and ECA109 cells (B).

FIG. 2 is a concentration inhibition rate curve of DDP on different ESCC cells, A is a concentration inhibition rate curve of DDP on KYSE450/DDP and KYSE450 cells, and B is a concentration inhibition rate curve of DDP on ECA109/DDP and ECA109 cells.

FIG. 3 shows the results of the radiation clone formation experiment.

FIG. 4 is a concentration inhibition rate curve of ESCC DDP-resistant cell lines treated with different concentrations of DDP combined with fingolimod or DMSO, A is KYSE450/DDP cells, and B is ECA109 cells.

FIG5 is a gel electrophoresis diagram of the DARTS experiment screening fingolimod's anti-esophageal squamous cell carcinoma target.

DETAILED DESCRIPTION

The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

Example 1: CCK8 method to detect the inhibitory effect of fingolimod on human esophageal squamous cell carcinoma cells (IC ₅₀ )

Tumor cells (KYSE450 cells and ECA109 cells) in the logarithmic growth phase were counted and prepared into a cell suspension with a concentration of 6×10 ⁴ cells/mL. 100 μL was added to a 96-well plate and placed in a 37°C, 5% CO ₂ cell culture incubator for overnight culture. After the cells adhered to the wall, different concentrations of fingolimod were added, with a concentration gradient of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 4 μM, 8 μM, and 10 μM. Three replicates were set for each group, and the drug solvent was used as the control group. After 48 hours of culture, 10 μL of CCK-8 working solution was added, and the cells were incubated in the cell culture incubator for another 3 hours. The OD value was detected at 450 nm and calculated.

The results are shown in Figure 2. Fingolimod can effectively inhibit the growth of KYSE450 and ECA109 cells and reduce cell viability, with _IC50 values of 50.23nM and 72.28nM, respectively.

Example 2: Construction of cisplatin-resistant human esophageal squamous cell carcinoma cell line

Esophageal squamous cell carcinoma (ESCC) cell lines KYSE450 and ECA109, which are sensitive to cisplatin (DDP, with a small IC ₅₀ value), were selected, and KYSE450/DDP and ECA109/DDP were established by high-concentration shock combined with low-concentration continuous induction. The specific method is as follows: cells in the logarithmic growth phase were treated with a medium containing 25μg/mL DDP for 2h, washed with PBS three times to remove dead cells, and replaced with a medium containing IC ₅₀ concentration of DDP for 10-14 days until the cells grew stably and were continuously passaged for 5 times, and the cell IC ₅₀ value was determined again. Then, the medium containing 25μg/mL DDP was used again for 2h until the cells could grow normally in the medium containing 25μg/mL DDP, and they were named KYSE450/DDP and ECA109/DDP. To ensure the stability of the cell resistance to DDP, the medium containing 25μg/mL DDP was used for culture for one week every month thereafter.

CCK8 method to detect cell resistance, CCK8 detection steps:

This was done using the CCK-8 cell proliferation kit from Shanghai Taoshu Biotechnology Co., Ltd.

(1) Inoculate the cell suspension into a 96-well plate, 100 μL per well, 2,000 cells per well.

(2) Culture or administer drugs for an appropriate period of time according to experimental needs.

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

(4) The absorbance value was measured by the ELISA instrument at a wavelength of 450 nm, the inhibition rate at different concentrations was calculated, and the concentration inhibition rate curve was drawn.

The results are shown in Figure 2. The inhibition rate and IC ₅₀ of DDP on KYSE450/DDP and ECA109/DDP cell lines were significantly lower than those of KYSE450 and ECA109, which met the evaluation requirements for drug-resistant strains.

Example 3: Fingolimod increases the sensitivity of drug-resistant strains to radiation

The radiation clone formation experiment was used to determine the effect of fingolimod on the radiation sensitivity of drug-resistant strains. The specific steps are as follows:

(1) ESCC resistant cell lines KYSE450/DDP and ECA109/DDP were placed in 1640 medium containing 10% fetal bovine serum. 10 μM fingolimod was added to the experimental group, while no fingolimod was added to the control group. After irradiation with 4 Gy of X-ray, the cells in different groups were cultured normally for 24 h.

(2) Adjust the cell concentration to 1×10 ³ cells/ml, take 200 μl and inoculate it into a 6 cm culture dish, and add 10 ml of culture medium.

(3) Cultivate for 2 weeks and stop culturing when colonies are visible to the naked eye.

(4) Discard the culture medium and wash twice with PBS.

(5) Fix with 5 ml of anhydrous methanol for 15 min and discard the fixative.

(6) Add 0.1% crystal violet stain for 20 min, rinse with tap water, and air dry.

(7) Take photos and count the number of colonies visible to the naked eye. Calculate the clone formation rate = number of colonies/number of inoculated cells.

The results are shown in Figure 3. Compared with X-ray irradiation alone, the clone numbers of the resistant cell lines KYSE450/DDP and ECA109/DDP were significantly reduced in the combination of fingolimod and X-ray irradiation. The results showed that fingolimod treatment can increase the radiation sensitivity of ESCC DDP-resistant cell lines and reduce the radiotherapy resistance of ESCC DDP-resistant cell lines.

Example 4: Fingolimod increases the sensitivity of drug-resistant strains to cisplatin

The ESCC resistant cell lines KYSE450/DDP and ECA109/DDP were treated with cisplatin and fingolimod or DMSO. The IC ₅₀ values were detected by CCK8 assay to observe the sensitivity of fingolimod-resistant cell lines to cisplatin.

The results are shown in Figure 4. As the concentration increases, the inhibitory effect of DDP on ESCC resistant cell lines increases. At different concentrations, the inhibition rates of KYSE450/DDP and ECA109/DDP treated with DDP combined with fingolimod were significantly higher than those of DDP combined with DMSO, and the IC ₅₀ value of fingolimod was 0.032 μM, and the IC ₅₀ value of DMSO was 0.32 μM. The IC ₅₀ value of fingolimod added to the ECA109/DDP resistant strain was 0.32 μM, and the IC ₅₀ value of DMSO added was 3.2 μM. The results show that fingolimod treatment can increase the chemotherapy sensitivity of ESCC DDP-resistant cell lines and reduce the chemotherapy resistance of ESCC DDP-resistant cell lines.

Example 5: DARTS experiment to screen targets of fingolimod

Drug Affinity Response Targeted Stability Technology (DARTS) is a method for screening drug targets based on the assumption that the binding of drugs to target proteins may render the target proteins resistant to protease hydrolysis.

1) Fingolimod was co-incubated with protein samples of KYSE450/DDP and ECA109/DDP-resistant cells;

2) Add M-PER lysis buffer containing protease inhibitors and phosphatase inhibitors, and collect cells (at least 8×10 ⁸ cells) using a cell scraper;

3) Gel electrophoresis and staining: the experimental group may have specific bands compared with the control group;

4) Mass spectrometry identification of differential bands.

The results are shown in Table 1 and Figure 5. Compared with the control group (solvent group), 7 specific peptides were detected in the cells of the fingolimod-treated group, and the ratio of the specific peptides in the drug group and the control group was 6.06213. The specific peptide was a unique peptide of NUP54 (P<0.05).

Table 1 Targets of fingolimod

Gene name Specific peptides The ratio of drug group to control group P-value NUP54 7 6.08213 0.00326

It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (10)

1. Use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs, characterized in that the cancer includes esophageal squamous cell carcinoma. 2. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 1, characterized in that the drug is used to promote cancer cell apoptosis, reduce cancer cell activity, inhibit cancer cell proliferation, and curb cancer cell migration. 3. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 1, characterized in that the use is the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for improving the chemotherapy sensitivity of esophageal squamous cell carcinoma. 4. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 3, characterized in that the chemotherapy drug includes cisplatin. 5. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 1, characterized in that the use is the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for improving the radiosensitivity of esophageal squamous cell carcinoma. 6. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 1, characterized in that the use is the use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof combined with cisplatin in the preparation of esophageal squamous cell carcinoma therapeutic drugs. 7. The use of fingolimod, its derivatives or pharmaceutically acceptable salts thereof in the preparation of cancer therapeutic drugs according to claim 6, characterized in that the fingolimod, its derivatives or pharmaceutically acceptable salts thereof and cisplatin are administered simultaneously or continuously in any order. 8. A cancer treatment drug, characterized in that the active ingredients include cisplatin and fingolimod, its derivatives or pharmaceutically acceptable salts thereof as described in claim 1. 9. The drug according to claim 8, characterized in that it further comprises a pharmaceutically acceptable excipient. 10. The drug according to claim 9, characterized in that the auxiliary material comprises a diluent, a filler, a binder, a disintegrant, a lubricant, a wetting agent, a stabilizer, an osmotic pressure regulator or a preservative.