Application of ramatroban in preparation of medicine for preventing or treating radioactive esophageal injury caused by ionizing radiation

Abstract

The invention relates to the technical field of medicine, ramatroban is innovatively applied to the protection direction of ionizing radiation damage, and research finds that on the cell and organoid level, through ramatroban pretreatment, the morphological recovery and proliferation of cells can be remarkably promoted, the cell activity can be improved, the release of lactic dehydrogenase can be reduced, and the purpose of preventing and treating the ionizing radiation damage can be achieved. The cell apoptosis rate is reduced. Meanwhile, the proliferation speed of the organoid is increased, the volume and shape of the organoid are improved, and the expression of inflammatory factors is reduced, which proves that the ramatroban has potential application value and development prospect in the aspect of preventing and treating the ionizing radiation injury.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to the application of ramatroban in the preparation of a drug for preventing or treating radiation-induced radioactive esophageal injury. Background Art

[0002] Radiation-induced esophageal injury is one of the most common complications during radiotherapy or chemoradiotherapy for head and neck tumors, lung cancer, esophageal cancer, mediastinal tumors, etc. Radiation-induced esophageal injury can cause exudative changes such as congestion, edema, and erosion of the esophageal mucosa, resulting in ulcers, perforations, strictures, and obstructions, leading to symptoms such as dysphagia, difficulty in eating, or burning discomfort behind the sternum in patients, and even suspension of radiotherapy, which is an important factor restricting the radiotherapy effect. Although studies have reported that EGCG, honey, amifostine, etc. can relieve the symptoms of patients, there is still a lack of effective prevention and treatment measures clinically. Therefore, there is an urgent need to develop a drug for radiation-induced esophageal injury to improve the current situation of high limitations in clinical medication and provide new ideas for the clinical prevention and treatment of radiation-induced esophageal injury.

[0003] Ramatroban is a selective thromboxane A2 (TxA2) and PGD2 antagonist, which mainly regulates the immune response and inflammatory process by inhibiting the action of PGD2 in vivo. PGD2 is a lipid mediator produced by immune cells such as mast cells and eosinophils, and is involved in the pathogenesis of various immune-related diseases. Both PGD2 and TXA2 were significantly upregulated in the lungs of human patients and mice with silicosis, and ramatroban significantly alleviated silica-induced pulmonary inflammation, fibrosis, and cardiopulmonary dysfunction. In multiple studies on asthma and allergic diseases, it was found that ramatroban could significantly reduce the allergic reactions mediated by PGD2 and relieve symptoms such as nasal congestion and runny nose. Ramatroban could also significantly inhibit the following ovalbumin-induced allergic characteristics: sneezing, nasal rubbing, eosinophil infiltration, IL-16 expression in nasal tissues, and serum IL-16 levels. In addition, Xue et al. found that PGD2 preferentially upregulates the production of pro-inflammatory cytokines in human Th2 cells through a CRTH2-dependent mechanism, while ramatroban can significantly inhibit PGD2-induced Th2 cytokine production. However, currently, there is no report on the application of ramatroban in the inflammatory response caused by radiation injury. Summary of the Invention

[0004] In view of this, the present invention provides the application of ramatroban in the preparation of a drug for preventing or treating radiation-induced radioactive esophageal injury.

[0005] The specific technical solution of the invention is as follows:

[0006] The ramatroban system of the present invention is named 3,4-dihydro-1H-carbazol-9(2H)-yl)propanoic acid, which is a selective thromboxane A2 (TxA2) antagonist; the CAS registration number is 116649-85-5; the chemical formula is C 21 H 21 FN2O4S; the molecular weight is 416.47; the appearance is a white to off-white solid; the solubility is soluble in ethanol, DMSO and other organic solvents, and has limited solubility in water, belonging to a drug with low water solubility.

[0007] What the present invention aims to protect includes:

[0008] The use of ramatroban in the preparation of a drug for preventing or treating radiation-induced radioactive esophageal injury.

[0009] The use of ramatroban in the preparation of a drug for reducing the inflammation of radiation-induced radioactive esophageal injury.

[0010] The use of ramatroban in the preparation of an anti-radiation drug.

[0011] Experimental data show that at the cell and organoid levels, after pretreatment with ramatroban, it can significantly promote the morphological recovery and proliferation of cells, improve cell viability, reduce the release of lactate dehydrogenase (LDH), and decrease the apoptosis rate. At the same time, the proliferation rate of organoids is accelerated, the volume and shape are improved, and the expression of inflammatory factors is downregulated. It proves that ramatroban has potential application value and development prospects in the prevention and treatment of radiation injury.

[0012] The use of ramatroban in the construction of a cell model of radioactive esophageal injury.

[0013] The use of ramatroban in the construction of an organoid model of radioactive esophageal injury.

[0014] The use of a pharmaceutical composition in the preparation of a drug for preventing or treating radiation-induced radioactive esophageal injury, wherein the pharmaceutical composition comprises a therapeutically effective amount of ramatroban and a pharmaceutically acceptable excipient.

[0015] The use of a pharmaceutical composition in the preparation of a drug for reducing the inflammation of radiation-induced radioactive esophageal injury, wherein the pharmaceutical composition comprises a therapeutically effective amount of ramatroban and a pharmaceutically acceptable excipient.

[0016] The use of a pharmaceutical composition in the preparation of an anti-radiation drug, wherein the pharmaceutical combination comprises a therapeutically effective amount of ramatroban and a pharmaceutically acceptable excipient.

[0017] Furthermore, pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweetening agents, and pigments.

[0018] Furthermore, the pharmaceutical composition is a liquid preparation, a solid preparation or a spray preparation.

[0019] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0020] 1. The present invention innovatively applies rematriptan to the field of protection against X-ray ionizing radiation damage. Experimental data show that at the cellular and organoid levels, pretreatment with rematriptan can significantly promote the morphological recovery and proliferation of cells, improve cell viability, reduce the release of lactate dehydrogenase (LDH), and decrease the apoptosis rate. At the same time, the proliferation rate of organoids is accelerated, the volume and shape are improved, and the expression of inflammatory factors is downregulated. This demonstrates the potential application value and development prospects of rematriptan in the prevention and treatment of ionizing radiation damage.

[0021] 2. The present invention proposes the application of rematriptan in the construction of a cell model of radioactive esophageal injury. Using human normal esophageal epithelial cells HEEC and Het-1A as research objects, HEEC cells irradiated with 0, 4, 8 Gy were pretreated with 0, 70 μM rematriptan, and Het-1A cells irradiated with 0, 4, 8 Gy were pretreated with 0, 80 μM rematriptan. By detecting a variety of cell phenotype experiments respectively, it was found that after incubation with rematriptan, the irradiated HEEC and Het-1A cells showed morphological recovery, increased proliferation rate, enhanced activity, less LDH release, and reduced apoptosis, showing a mitigating effect in the construction of the cell model.

[0022] 3. The present invention proposes the application of rematriptan in the construction of an organoid model of radioactive esophageal injury. Using human esophageal organoids as research objects, human esophageal organoids irradiated with 0, 2 Gy were pretreated with 0, 30 μM rematriptan. By detecting the organoid formation ability, the morphology and average area statistics of organoids, and the mRNA expression of esophageal inflammatory factors TNF-α, IFN-γ and Cox2, it was found that after incubation with 30 μM rematriptan, the irradiated human esophageal organoids had an accelerated proliferation rate, larger organoid size and shape, and reduced expression of inflammatory factors, showing a mitigating effect in the construction of the organoid model. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram for observing the cell morphology of rematriptan.

[0024] Figure 2 It is a diagram for measuring the cell viability of rematriptan.

[0025] Figure 3 It is a diagram for measuring the lactate dehydrogenase (LDH) of rematriptan cells.

[0026] Figure 4 It is a diagram for detecting the apoptosis of rematriptan cells.

[0027] Figure 5 It is a schematic diagram for detecting the growth of esophageal organoids and inflammatory factors of rematriptan. Detailed implementation manners

[0028] The present invention will be described in detail below with reference to the accompanying drawings.

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The embodiments of the present application will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, but there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where the detailed descriptions of well-known matters are omitted and the repeated descriptions of actually identical structures are omitted. This is to avoid the following descriptions from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following descriptions are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0031] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs; the terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application; unless otherwise stated, the numerical values of the various parameters mentioned in the present application can be measured by various commonly used measurement methods in the art (for example, they can be tested according to the methods given in the embodiments of the present application).

[0032] The terms "including" and "having" and any variations thereof in the description and claims of the present application are open expressions, that is, they include the content specified in the present application, but do not exclude other aspects.

[0033] In the description of the present application, "A and / or B" may include the case of A alone, the case of B alone, and any one of the cases of A and B, where A and B are only for example and may be any technical features connected by "and / or" in the present application.

[0034] If there is no special description, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0035] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form new technical solutions.

[0036] In the following examples, the ramatroban system used is 3,4-dihydro-1H-carbazol-9(2H)-yl)propanoic acid, which is a selective thromboxane A2 (TxA2) antagonist; the CAS registration number is 116649-85-5; the chemical formula is C 21 H 21 FN2O4S; the molecular weight is 416.47; the appearance is white to off-white solid; the solubility is soluble in ethanol, DMSO and other organic solvents, and has limited solubility in water, belonging to a drug with low water solubility.

[0037] In the following examples, ramatroban was purchased from Bidepharm, with the product number: BD89188 and the CAS number: 116649-85-5.

[0038] Example 1

[0039] Observation of cell morphology

[0040] Suspend the well-growing human normal esophageal epithelial cells HEEC and Het-1A cells, and inoculate them into 6-well plates at 4×10 4 cells / well, divided into 4Gy and 8Gy irradiation groups and a control group (0Gy), placed in an incubator at 37°C and 5% CO2. When the cell confluence in each well is about 50%, carefully aspirate the culture medium, wash twice with PBS, then add ramatroban at concentrations of 0.0000 μmol / mL and 70.0000 μmol / mL to HEEC cells; add ramatroban at concentrations of 0.0000 μmol / mL and 80.0000 μmol / mL to Het-1A cells. There are 3 parallel wells in each group, and continue to culture in the incubator for 24h. Then place the cells in the irradiation group in the biological X-ray irradiator KUBTEC XCELL 320, with a dose rate of 1.7Gy / min and irradiation doses of 4Gy and 8Gy. The control group is sham-irradiated (bring the cell culture plate to the irradiation room but not irradiate). After irradiation, continue to observe the cell morphology under the microscope and take pictures, and stop observing 48h after irradiation.

[0041] As Figure 1 shown. After ramatroban intervention, the cell morphology of irradiated HEEC and Het-1A cells recovered and the proliferation rate became faster.

[0042] Example 2

[0043] Cell viability assay

[0044] Suspend the well-growing normal human esophageal epithelial cells HEEC and Het-1A cells, and inoculate them into 96-well plates at a system of 100 μL per well (0.4×10 4 cells / well). Divide them into 4 Gy and 8 Gy irradiation groups and a control group (0 Gy), and place them in an incubator at 37 °C and 5% CO2 for culture. When the cell confluence in each well is about 60%, carefully aspirate the culture medium. After washing twice with PBS, add rematriptan at concentrations of 0.0000 μmol / mL and 70.0000 μmol / mL to HEEC cells; add rematriptan at concentrations of 0.0000 μmol / mL and 80.0000 μmol / mL to Het-1A cells. There are 6 parallel wells in each group. After continuing to culture in the incubator for 24 h, place the cells in the irradiation group in a biological X-ray irradiator KUBTEC XCELL 320 with a dose rate of 1.7 Gy / min and irradiation doses of 4 Gy and 8 Gy. The control group is subjected to sham irradiation (bring the cell culture plate to the irradiation room but do not irradiate). At 24 h and 48 h after irradiation, add 10 μL of CCK-8 solution to each well (in the dark), and continue to incubate in a 37 °C incubator for 1-2 h. Then, use an enzyme-labeled instrument to detect the absorbance (OD value) of each well at a wavelength of 450 nm, and calculate the cell viability based on the absorbance value.

[0045] The results are as Figure 2 shown. Rematriptan has no toxic effect on HEEC and Het-1A cells, and can relieve the decrease in cell viability caused by ionizing radiation in HEEC and Het-1A cells.

[0046] Example 3

[0047] Determination of cell lactate dehydrogenase (LDH)

[0048] Suspend the well-growing normal human esophageal epithelial cells HEEC and Het-1A cells, and inoculate them into 96-well plates at a system of 100 μL per well (0.3×10 4(cells / well), divided into 4 Gy and 8 Gy irradiation groups and a control group (0 Gy), placed in an incubator at 37 °C and 5% CO2. When the cell confluence in each well was about 60%, the culture medium was carefully aspirated. After washing twice with PBS, HEEC cells were added with rematriptan at concentrations of 0.0000 μmol / mL and 70.0000 μmol / mL; Het-1A cells were added with rematriptan at concentrations of 0.0000 μmol / mL and 80.0000 μmol / mL. There were 6 parallel wells in each group and they were placed in the incubator for further culture. After 24 h, the cells in the irradiation groups were placed in a biological X-ray irradiator KUBTEC XCELL 320 with a dose rate of 1.7 Gy / min and irradiation doses of 4 Gy and 8 Gy. The control group was sham-irradiated (the cell culture plates were brought to the irradiation room but not irradiated). More than 12 h before the detection point after irradiation, the culture medium in the well plates was replaced with serum-free medium and continued to be cultured until the detection point after irradiation for detection. When performing the detection, 10 μL of LDH release agent was added to the preset maximum enzyme activity control well and incubated at 37 °C for 1 h. The detection solution was prepared 10 min before the end of incubation, and the preparation ratio was 1 volume (enzyme solution): 1 volume (lactic acid): 1 (INT working solution). The INT working solution was prepared by mixing INT solution and INT diluent at a ratio of 1:9. After the incubation ended, 80 μL of the supernatant from each well was transferred to a new 96-well plate, 40 μL of the detection solution was added, and incubated in the dark at room temperature for 30 min. Subsequently, the values of each well were detected using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 490 nm.

[0049] The results are as Figure 3 shown. Rematriptan reduced the release of lactate dehydrogenase in HEEC and Het-1A cells caused by ionizing radiation.

[0050] Example 4

[0051] Cell apoptosis detection

[0052] Human normal esophageal epithelial cells HEEC and Het-1A cells in good growth state were resuspended and, according to 1×10 5Cells were seeded at a density of [number of cells] per well in a 6-well plate and divided into 4 Gy and 8 Gy irradiation groups and a control group (0 Gy). The plates were placed in an incubator at 37°C with 5% CO2. When the cell confluence in each well reached approximately 50%, the medium was carefully aspirated, and the cells were washed twice with PBS. Then, HEEC cells were treated with 0.0000 μmol / mL and 70.0000 μmol / mL of rematriptan, and Het-1A cells were treated with 0.0000 μmol / mL and 80.0000 μmol / mL of rematriptan. There were 3 parallel wells in each group. After continuing to culture in the incubator for 24 h, the cells in the irradiation groups were placed in a biological X-ray irradiator KUBTEC XCELL 320 with a dose rate of 1.7 Gy / min and irradiated at doses of 4 Gy and 8 Gy. The control group was sham-irradiated (the cell culture plates were brought to the irradiation room but not irradiated). At 24 h and 48 h after irradiation, the supernatants of each well were collected, the cell precipitates were digested and collected, and after washing twice with PBS, 200 μL / well of Annexin V / PI apoptosis detection kit binding buffer was added, mixed well, and then 5 μL of Annexin V-FITC and 10 μL of PI were added to each well. The cells were incubated at room temperature in the dark for 15 min. Subsequently, flow cytometry was used to detect the cell apoptosis level.

[0053] The results are as Figure 4 shown, and rematriptan reduced the apoptosis levels of HEEC and Het-1A cells induced by ionizing radiation.

[0054] Example 5

[0055] Rematriptan can significantly enhance the radiation resistance of normal human esophageal organoids.

[0056] The operation steps for culturing normal human esophageal organoids are as follows:

[0057] 1) The adjacent tissue of human esophageal squamous cell carcinoma freshly resected in the operating room was placed in ice-precooled Advanced DMEM / F12 medium supplemented with double antibiotics (1% vol / vol) and quickly transferred to the laboratory for subsequent operations.

[0058] 2) The washed esophageal tissue was minced and added to a digestive solution prepared from Collagenase Type Ⅰ (1 mg / ml), PBS, and Antibiotic-Antimycotic (1% vol / vol). The mixture was shaken at 37°C on a shaker at 100 rpm for 60 min and then centrifuged at 2000 rpm for 5 min.

[0059] 3) 5 mL of TrypLE TM Express was added to resuspend the precipitate, and the mixture was shaken at 37°C on a shaker at 100 rpm for 15 min.

[0060] 4) Neutralize TrypLE™ Express with an equal volume of Advanced DMEM / F12 containing FBS (10% vol / vol) to terminate digestion. Filter the tissue suspension through a 70-μm filter twice and centrifuge at 2000 rpm for 5 min.

[0061] 5) Add pre-chilled DPBS and wash 1 - 2 times, then centrifuge at 2000 rpm for 5 min.

[0062] 6) Discard the supernatant, add Matrigel to resuspend the precipitated cells on ice, try to avoid air bubbles, and seed the cells at 50 μm / well in a low-attachment 24-well culture plate. Place it in the incubator and let it stand for 15 min.

[0063] 7) Add 500 μL of pre-warmed human esophageal organoid medium to each well, and then culture in a cell culture incubator at 37°C and 5% CO2. Replace the medium every 2 - 3 days. Organoids can be observed after 5 - 7 days of culture.

[0064] 8) Digest and resuspend the successfully cultured P3-generation human esophageal organoids, and seed them at 0.5×10 4 cells / well in a low-attachment 24-well plate. Divide them into a 2-Gy irradiation group and a 0-Gy control group, and place them in an incubator at 37°C and 5% CO2. When the cell confluence in each well reaches about 60%, carefully aspirate the medium, wash twice with PBS, and then add 0.0000 μmol / mL and 30.0000 μmol / mL of ramatroban to the normal human esophageal organoids. There are 3 parallel wells in each group. Continue to culture in the incubator for 24 h, then place the irradiated organoids in a biological X-ray irradiator KUBTEC XCELL 320 with a dose rate of 1.7 Gy / min and a dose of 2 Gy. The control group is subjected to sham irradiation (bring the organoid culture plate to the irradiation room but do not irradiate).

[0065] 9) Observe and photograph the morphological changes of the cell spheres under a microscope at 2, 4, and 6 days after irradiation. Extract the organoid RNA on the 6th day after irradiation for QT-qPCR experiments to detect the expression levels of esophageal inflammatory factors. The specific primer sequences are as follows:

[0066] GAPDH-F: 5′-TCCTCCACCTTTGACGCT-3′.

[0067] GAPDH-R: 5′-CCACCACCCTGTTGCTGT-3′.

[0068] TNF-α-F: 5′-GCCTCGCCCTTTGCTTTACT-3′.

[0069] TNF-α-R: 5′-CTGTGGGTCTCAGGGAGATCA-3′.

[0070] IFN-γ-F: 5'-TCGGTAACTGACTTGAATGTCCA-3'.

[0071] IFN-γ-R: 5'-TCGCTTCCCTGTTTTAGCTGC-3'.

[0072] Cox2-F: 5'-CTGGCGCTCAGCCATACAG-3'.

[0073] Cox2-R: 5'-CGCACTTATACTGGTCAAATCCC-3'.

[0074] The results are as Figure 5 shown. After incubation with 30 μM of rematriptan, the proliferation rate of irradiated human esophageal organoids became faster, the shape and size of the organoids were larger, and the expression of inflammatory factors decreased, indicating that rematriptan can enhance the anti-radiation ability of normal human esophageal organoids.

[0075] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The use of Ramatroban in the preparation of drugs for preventing or treating radiation-induced esophageal injury caused by ionizing radiation.

2. The use of Ramaquban in the preparation of drugs for reducing inflammation of radiation-induced esophageal injury caused by ionizing radiation.

3. Application of Ramaquban in the preparation of anti-radiation drugs.

4. Application of Ramaquilla in constructing a cell model of radiation-induced esophageal injury.

5. Application of Ramaquilla in constructing an organoid model of radiation-induced esophageal injury.

6. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating radiation-induced esophageal damage caused by ionizing radiation, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of ramatroban and pharmaceutically acceptable excipients.

7. Use of a pharmaceutical composition in the preparation of a drug for alleviating radiation-induced esophageal injury and inflammation, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of ramatroban and pharmaceutically acceptable excipients.

8. Use of a pharmaceutical composition in the preparation of an anti-radiation drug, characterized in that: The drug combination comprises a therapeutically effective amount of ramatroban and pharmaceutically acceptable excipients.

9. The use according to any one of claims 6 to 8, characterized in that: Pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and pigments.

10. The use according to any one of claims 6 to 8, characterized in that: The pharmaceutical composition is a liquid preparation, a solid preparation or a spray preparation.

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