CN115645532B - Application of isoxazole derivative in preparing brain glioma radiotherapy sensitization drugs - Google Patents
Application of isoxazole derivative in preparing brain glioma radiotherapy sensitization drugs Download PDFInfo
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Abstract
The invention relates to the field of medicines, in particular to a novel application of an isoxazole derivative, namely an application in preparing a brain glioma radiotherapy sensitization medicine, wherein the structural formula of the isoxazole derivative is shown as follows. The isoxazole derivative can be used for preparing a radiotherapy sensitization medicine, and can obviously increase the radiotherapy sensitivity of brain glioma cells under the concentration without toxic or side effect
。
Description
Technical Field
The invention relates to the field of medicines, in particular to a new application of an isoxazole derivative, namely an application in preparing a radiotherapy sensitization medicine.
Background
Glioblastoma is the most common primary intracranial malignant tumor of adults, and has the characteristics of high invasiveness and high death rate. The current standard treatment for glioblastoma includes maximum surgical resection, postoperative radiation therapy, and temozolomide concurrent and adjuvant chemotherapy. Despite the combination therapy, glioblastoma patients had very poor prognosis, with a median overall survival of only 12-16 months. And almost all patients have relapsed or progressed within a short time after initial treatment, while the life expectancy after tumor recurrence is reduced to 5-10 months. Radiation therapy is an important treatment not alternative to glioblastoma, whether at initial diagnosis or after recurrence.
Rays are a moving element that may have kinetic energy, mass, charge, new elements, etc. characteristics. Radiation therapy has been applied to tumors for many years and is recommended for more than half of malignant patients. Examples of the radiation used for treating cancer include X-rays, proton beams, electron beams, heavy ion beams, and the like. Electromagnetic radiation such as X-rays and gamma rays is the current conventional radiotherapy means.
The main mechanism of X-ray treatment of cancer is to induce cell DNA damage, inhibit cancer cell proliferation, induce apoptosis, etc. However, since the X-rays are not selective for cancer cells and normal cells in the irradiated area, they damage normal tissues and cells while killing tumor cells. Meanwhile, the tumor cells have a set of efficient and accurate DNA damage repair system, and the tumor cells recover the cell activity through the DNA damage repair system, which is a main reason for poor tumor radiotherapy effect. In addition, gliomas are less sensitive to radiation. Therefore, how to improve the therapeutic effect of tumor cells with strong resistance to X-ray radiation is a problem to be solved. Certain chemical drug treatments are given to patients to improve the radiation sensitivity of tumor cells to the radiation, which is an important means for improving the tumor treatment effect of the radiation; such chemicals are commonly referred to as radiosensitizers.
The isoxazole derivative is a high-efficiency inhibitor of heat shock protein 90 (HSP 90). HSP90 is a chaperone protein that has the effects of regulating the posttranslational folding, stability and physiological function of its client proteins. HSP90 has a number of client proteins including EGFR, c-MET, BCR-ABL, B-RAF, c-KIT and HER2, etc. [ Ganetespib and HSP90: translating preclinical hypotheses into clinical promise ]. There is no document or patent reporting the radiotherapy sensitization effect of the isoxazole derivatives.
Disclosure of Invention
The invention aims to provide a new application of an isoxazole derivative, namely an application in preparing a brain glioma radiotherapy sensitization medicine.
In order to achieve the above object, the first aspect of the present invention provides an application of an isoxazole derivative in preparing a brain glioma radiotherapy sensitization drug, wherein the isoxazole derivative has a structural formula as shown in formula I below:
formula I.
Preferably, the radiotherapy sensitization medicine is a radiotherapy sensitization medicine suitable for brain glioma.
Preferably, the radiotherapy sensitization medicine is an oral preparation or an injection preparation.
Preferably, the administration time of the radiotherapy sensitization medicine is 1 hour to 0 hour before irradiation.
Preferably, the radiotherapy sensitization medicine is used in vitro with an effective dose of between 10 and 100 and nM.
Preferably, the effective dose of the radiotherapy sensitization medicine is between 5 and 50 mg/kg BIW in vivo.
According to the invention, isoxazole derivatives with different concentrations are directly added to brain glioma cells T98G for 24 hours and 48 hours, and then CCK-8 is adopted to detect cell growth and proliferation, so that the results show that: the isoxazole derivatives at a concentration of 5nM or less have a slight inhibition of T98G cells, and the inhibition rates are within 5%.
Meanwhile, the invention compares the difference of the proliferation of T98G cells after the X-ray irradiation and the X-ray irradiation plus isoxazole derivative treatment for 24 hours and 48 hours, and the result shows that: the proliferation of T98G cells can be obviously inhibited by using X-ray irradiation after pretreatment of isoxazole derivatives (5 nM) with no toxic or side effect concentration. In addition, after T98G cells are treated by 5nM isoxazole derivatives for 1 hour to 0 hour, 4Gy of X-ray irradiation is further given to the cells, and then after the cells are continuously cultured for 48 hours, apoptosis is detected by a flow cytometer after double-dyeing by Annexin V-FITC and PI, and the result shows that: the pretreatment of isoxazole derivatives (5 nM) with no toxic or side effect concentration can significantly increase the apoptosis rate of T98G cells induced by radiotherapy. These study data indicate that isoxazole derivatives can increase the radiosensitivity of brain glioma cells.
In addition, female C57BL/6 mice of 6 weeks old purchased by the national academy of sciences laboratory animal center are used for animal experiments. Mice were fed in cages with daily pad changes at 25±1 ℃. All mice were implanted with 5 x 10≡6T 98G cells (100 μl PBS) at a fixed location (subcutaneously in the hind leg). After tumor formation, the treated mice were randomly divided into 3 groups: group 1, non-irradiated + saline control group; group 2, irradiation+saline group; group 3, irradiation+isoxazole derivative group. The tumor was locally irradiated at the time of irradiation, and the isoxazole derivative was administered by intravenous injection at 12.5mg/kg TIW 1 hour to 0 hour before irradiation. Then continuously feeding tumor-bearing mice to observe the curative effect, and taking out the tumor to measure the size. The results show that the average diameter of the tumors of the mice which are not irradiated and treated by the isoxazole derivatives is the largest, the average diameter of the tumors of the mice in the irradiated group is 5mm smaller than that of the mice in the control group, and the average diameter of the tumors of the mice in the irradiated and isoxazole derivative combined treatment group is 10mm smaller than that of the mice in the control group, so that the isoxazole derivatives have obvious radiosensitization effect.
Therefore, the invention claims the application of the isoxazole derivative in preparing brain glioma radiotherapy sensitization drugs. The isoxazole derivative with no toxic or side effect concentration is adopted to pretreat the glioma cells, so that the sensitivity of radiotherapy can be improved, and the curative effect of the radiotherapy can be improved.
The invention has the beneficial effects that:
the isoxazole derivative can be used for preparing a radiotherapy sensitization medicine, and can obviously increase the radiotherapy sensitivity of brain glioma cells under the concentration of no toxic or side effect. And the effect of single irradiation in X-ray treatment can be improved, thereby being beneficial to reducing the treatment times, shortening the treatment course and reducing the treatment cost.
Drawings
FIG. 1 shows the effect of different doses of X-rays on T98G cell growth and proliferation in the examples.
FIG. 2 is a graph showing the effect of various concentrations of isoxazole derivatives on T98G cell growth and proliferation in the examples.
FIG. 3 is a graph showing the effect of the combined use of isoxazole derivatives and X-rays on T98G cell proliferation in the examples.
FIG. 4 is a graph showing the effect of the combined use of isoxazole derivatives and X-rays on T98G apoptosis in the examples.
FIG. 5 is a graph of the radiosensitization effect of isoxazole derivatives in the examples.
Detailed Description
The following provides a detailed description of specific embodiments of the invention with reference to the examples and figures.
Materials:
cell lines and cell culture: human glioma cell line T98G cells were cultured in 1640 medium containing 10% fetal bovine serum, 1% glutamate, and antibiotics (penicillin plus streptomycin). The cells were cultured in an incubator at 37℃with 5% CO2, and transferred every 2-3 days for one generation, and the cells in logarithmic growth phase were taken for the experiment.
The medicine and the main reagent are the isoxazole derivative, which is dissolved in PBS to prepare mother liquor with the concentration of 10mmol/L, the mother liquor is preserved in a refrigerator with the temperature of 4 ℃, and the mother liquor is prepared from RMPI 1640 culture medium, fetal calf serum, pancreatin, 4% paraformaldehyde, 1% crystal violet solution, CCK-8 reagent, annexin V-FITC and PI.
Example 1: effect of X-ray irradiation on T98G cell clone formation.
Cell culture: T98G cells were cultured in 1640 medium containing 10% fetal bovine serum. All cells were cultured in a 5% CO2 incubator at 37℃and passaged every 2-3 days, and cells in logarithmic growth phase were taken for the experiment.
Cell clone formation method: taking T98G cells in a logarithmic growth phase, and giving X-ray radiation with different dosages (0 Gy, 2Gy, 4Gy, 6Gy and 8 Gy); cells were digested with trypsin 24h after irradiation, and cells were harvested and counted after termination of digestion. Cells were seeded in groups of 1000 cells per well in a ratio of 2mL medium in 12-well plates. Each group is provided with 3 parallels. After about 10-14 days, the culture can be terminated after the cells form clonal colonies. The residual cell culture medium was washed off with PBS, and 2ml of 4% paraformaldehyde was used to fix the cells at room temperature for 15min. The cells were then washed with PBS, followed by staining with 1% crystal violet solution at room temperature for 20min. After the dyeing is finished, the crystal violet solution is gently flushed with running water and dried in a fume hood. Clones of more than 50 cell compositions were counted under an optical microscope. Cell viability scores (Survival Fraction, SF) were determined according to equations 1 and 2, while nonlinear curve fitting was performed on the data using Origin software. Equation 1: sf=number of colonies formed/(number of cells seeded) PE. Equation 2: PE = number of colonies formed by control/number of cell inoculations of control.
The results are shown in figure 1, and show that the X-ray irradiation has obvious inhibition effect on the growth and proliferation of T98G cells, the single-click multi-target model is adopted to perform data fitting on the survival scores of the cells, the average lethal dose is 2.7Gy, the quasi-threshold dose is 4.3Gy, and therefore, the irradiation dose of 4Gy is selected for the next experiment.
Example 2: toxicity test of isoxazole derivatives on tumor cells;
cell culture was as in example 1.
CCK-8 (Cell Counting Kit-8) method: cells in the logarithmic growth phase were taken, and cells were collected and counted after regular digestion with pancreatin. The desired cell suspension was prepared in a ratio of 5000 cells per well of 200 μl medium, and the cell suspension was inoculated into 96-well plates with a row gun, 5 in parallel per group. After cell attachment, the blank medium was changed to medium containing different concentrations of isoxazole derivatives (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 nM) and the culture was continued for 24h and 48h. The original medium was changed to a medium containing 10% CCK-8 reagent at a desired time point, and the culture was continued in an incubator for 0.5-3 hours, and the OD value of each group was measured at 450mM wavelength using an ELISA reader, and the cell viability was calculated according to formula 3. Cell viability was plotted. Equation 3: cell viability (%) = [ (OD dosing-OD blank)/(OD control-OD blank) ]x100%.
The results are shown in FIG. 2, and the results show that the isoxazole derivatives with the concentration of 1.25-5nM have smaller inhibition effect on T98G cells, and the inhibition rate is within 5%. Thus, this example selected 5nM isoxazole derivatives for the study of radiation sensitization-related aspects.
Example 3: isoxazole derivatives enhance X-ray induced cell proliferation inhibition;
cell culture was as in example 1.
CCK-8 is the same as in example 2. The experiments were divided into a control group, an isoxazole derivative group, an X-ray group, an isoxazole derivative+x-ray group. The isoxazole derivative concentration was 5nM and the X-ray irradiation dose was 4Gy. And (3) carrying out X-ray irradiation on the cells in the logarithmic growth phase, digesting the cells with trypsin 24h after the irradiation, and re-suspending the cells in a culture medium containing or not containing isoxazole derivatives according to grouping conditions to count the cells. Cells were seeded in 96-well plates at a ratio of 200 μl medium per well of 5000 cells, 5 in parallel per group. After 24h and 48h of culture, the culture medium is changed into a culture medium containing 10% of CCK-8 solution, and the culture is continued for 0.5-3h. The OD values of each group were measured with a microplate reader at 450mM wavelength and cell viability was calculated according to equation 3. Cell viability was plotted.
The result is shown in figure 3, the survival rate of the cells after the pure irradiation of X-rays for 24 hours is 91.7 percent, and the survival rate of the cells after the combined treatment of the X-ray irradiation and the isoxazole derivative for 24 hours is 61.6 percent; the survival rate of the cells after the pure irradiation of the X-rays for 48 hours is 86.8 percent, and the survival rate of the cells after the combined treatment of the X-ray irradiation and the isoxazole derivative for 48 hours is 48.7 percent. Studies have demonstrated that isoxazole derivatives have radiosensitization effects on T98G cells.
Example 4: isoxazole derivatives enhance X-ray induced apoptosis;
cell culture was as in example 1.
Flow cytometry to detect apoptosis: the influence of isoxazole derivatives combined with X-rays on apoptosis is detected by an Annexin V-FITC/PI double-dyeing apoptosis detection method. The experiments were divided into a control group, an isoxazole derivative group, an X-ray group, an isoxazole derivative+x-ray group. Cells of logarithmic growth phase were taken and planted in 60mm dishes. After the cells adhere to the wall, X-ray irradiation is performed. After 24h of irradiation, the cell culture medium was changed to fresh medium with or without isoxazole derivatives according to the grouping situation, and after further culturing for 24h and 48h, the cells were digested with pancreatin without EDTA, centrifuged at 800rpm for 4 min, and washed twice with PBS. Washing the supernatant, suspending the cells in 100 μl of 1 Xbuffer, adding 5 μl of FITC and 5 μl of PI, incubating for 15min in dark, and detecting apoptosis with Flow Sight Flow cytometer; IDEAS software carries on apoptosis analysis; origin software plots.
The results are shown in fig. 4, and compared with a pure irradiation group, the isoxazole derivative is cooperated with the X-ray irradiation to improve the apoptosis rate of cells from 3.3% to 15.9%, so that the cells have obvious difference. Studies have demonstrated that isoxazole derivatives have radiosensitization effects on T98G cells.
Example 5: radiosensitization of isoxazole derivatives;
cell culture was as in example 1.
Mice were fed: mice are placed in a cage with padding daily replaced at 25+/-1 ℃ to ensure sufficient moisture and food.
Building and processing a tumor-bearing mouse model: taking T98G cells in logarithmic growth phase, centrifuging after pancreatin digestion, and blowing uniformly in PBS. All mice were implanted with 5 x 10≡6T 98G cells (100 μl PBS) at a fixed location (subcutaneously in the hind leg). After tumor formation, the treated mice were randomly divided into 3 groups: group 1, non-irradiated + saline control group; group 2, irradiation+saline group; group 3, irradiation+isoxazole derivative group. The tumor was locally irradiated at the time of irradiation, and the isoxazole derivative was administered by intravenous injection at 12.5mg/kg TIW 1 hour to 0 hour before irradiation. Then continuously feeding tumor-bearing mice to observe the curative effect, and taking out the tumor to measure the size.
The results are shown in fig. 5, and the average diameter of the tumors of the mice which are not irradiated and treated by the isoxazole derivatives is the largest, the average diameter of the tumors of the mice in the irradiated group is 5mm smaller than that of the mice in the control group, and the average diameter of the tumors of the mice in the irradiated and isoxazole derivative combined treatment group is 10mm smaller than that of the mice in the control group, so that the isoxazole derivatives have obvious radiosensitization effect.
Wherein, the irradiation conditions are as follows: the X-ray irradiation was performed on an X-ray apparatus attached to a fifth hospital at the university of middle mountain. The energy was 6MeV and the dose rate was 600cGy/min. And adding isoxazole derivatives with proper concentrations for treatment 1-0 hour before irradiation, culturing to different time points, and collecting samples for detection of different biological endpoints.
And (3) statistical treatment: all experiments in the above examples were repeated 3 more times and the results were expressed as mean ± variance. The relevant data were t-checked using SPSS statistical software, with significant differences at P < 0.05.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (5)
1. An application of an isoxazole derivative in preparing a radiotherapy sensitization medicine, wherein the radiotherapy is radiotherapy by using X-ray irradiation, the radiotherapy sensitization medicine is a radiotherapy sensitization medicine applicable to brain glioma, and the Chinese cultural name of the isoxazole derivative is: 3- (2, 4-dihydroxy-5-isopropylphenyl) -N-ethyl-4- (1, 2,3, 4-tetrahydroisoquinolin-6-yl) isoxazole-5-carboxamide; the structural formula of the isoxazole derivative is shown in the following formula I:
2. The use of an isoxazole derivative according to claim 1 for the preparation of a radiotherapy-sensitized drug, wherein the radiotherapy-sensitized drug is an oral preparation or an injection preparation.
3. The use of an isoxazole derivative according to claim 1 for the preparation of a radiotherapy-sensitized drug, wherein the radiotherapy-sensitized drug is used in vitro in an effective amount of between 10 and 100 nM.
4. The use of an isoxazole derivative according to claim 1 for the preparation of a radiotherapy-sensitized drug, wherein the radiotherapy-sensitized drug is used in vivo in an effective amount of between 5 and 50 mg/kg BIW.
5. The use of an isoxazole derivative according to claim 1 for the preparation of a radiation-sensitive drug, wherein the administration time of the radiation-sensitive drug is 1 hour to 0 hour before irradiation.
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