Application of combination of amlexanox and polydatin in preparation of preparation for preventing and / or treating radiation-induced lung injury

Abstract

The invention relates to application of amlexanox and polydatin in preparation of a preparation for preventing and / or treating radiation-induced lung injury, and belongs to the technical field of medicines. The invention provides application of amlexanox combined with polydatin in preparation of a medicine for preventing and / or treating radiation-induced lung injury. The amlexanox and the polydatin are combined for use, so that the radiation-induced lung injury caused by ionizing radiation can be well prevented and / or treated, and the survival rate of a radiation-induced lung injury model mouse is increased by more than 50% on the premise of remarkably reducing the administration dosage of the amlexanox and the polydatin; the traditional Chinese medicine composition has the characteristics of small dosage, excellent prevention and / or treatment effect, no toxic or side effect and no dose dependence.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of amlexanox combined with polydatin in the preparation of a preparation for preventing and / or treating radiation lung injury. Background Art

[0002] Radiotherapy is one of the clinical treatments for thoracic malignancies. During radiotherapy, lung tissue will inevitably be exposed to a certain dose of ionizing radiation, which can cause varying degrees of radiation-induced lung injury (RILI). RILI includes acute radiation pneumonitis (RP) and radiation-induced pulmonary fibrosis (RPF). RP, as an early manifestation, often occurs within 1 to 3 months after the start of radiotherapy, while RPF often occurs as a late event. The main clinical manifestations of RILI are inflammatory infiltration of the alveolar interstitium, progressive dyspnea, and deterioration of lung function, which can even lead to respiratory failure in severe cases, greatly reducing the treatment effect and prognosis survival rate of patients with malignant tumors.

[0003] Amifostine is the only radiotherapy protective agent approved for clinical use, but its obvious side effects such as hypotension, severe nausea and poor tolerance limit its wide clinical application. In addition, Nintedanib can be used to prevent RP and reduce the incidence of pulmonary fibrosis, but studies have found that it has the risk of causing adverse events such as abnormal liver function, hypertension, skin and subcutaneous tissue diseases, and nervous system diseases; Pentoxifylline combined with vitamin E can prevent RPF, but Pentoxifylline can cause vomiting, diarrhea, dizziness, headache and other adverse symptoms. Therefore, there is no trace of high efficiency, low toxicity or non-toxic, stable and effective drug for preventing and / or treating radiation lung injury in the prior art. Summary of the invention

[0004] The purpose of the present invention is to provide an application of amlexanox combined with polydatin in the preparation of a preparation for preventing and / or treating radiation-induced lung injury, so as to solve the problems of large dosage, more side effects, poor prevention effect and weak treatment stability in the prior art in the process of preventing and / or treating radiation-induced lung injury.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of amlexanox combined with polydatin in the preparation of a medicine for preventing and / or treating radiation lung injury.

[0007] Preferably, the radiation lung injury is lung injury caused by ionizing radiation.

[0008] Preferably, the radiation lung injury includes acute radiation pneumonitis or radiation pulmonary fibrosis.

[0009] Preferably, the mass ratio of amlexanol to polydatin is 2-12:15-45.

[0010] The present invention also provides a pharmaceutical composition for preventing and / or treating radiation-induced lung injury, the pharmaceutical composition comprising amlexanox, polydatin and a pharmaceutically acceptable carrier.

[0011] Preferably, the mass ratio of amlexanol to polydatin is 2-12:15-45.

[0012] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating radiation lung injury.

[0013] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating acute radiation pneumonitis.

[0014] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating radiation pulmonary fibrosis.

[0015] The present invention has the following technical effects and advantages:

[0016] The present invention combines amlexanox with polydatin, which can effectively prevent and treat radiation lung injury caused by ionizing radiation, and the dosage of amlexanox combined with polydatin is significantly lower than that of amlexanox or polydatin alone.

[0017] The invention combines amlexanox with polydatin to effectively treat radiation lung injury caused by ionizing radiation, and the combination of amlexanox and polydatin can increase the survival rate of radiation lung injury model mice by more than 50% under the premise of significantly reducing the dosage of amlexanox and polydatin, and more effectively inhibits the expression of TNF-α and the production and release of IL-6, and at the same time does not produce any toxic side effects on normal alveolar epithelial cells, and has the advantages of small dosage, excellent therapeutic effect, no toxic side effects, and no dose dependence.

[0018] The present invention combines amifostine with polydatin to prevent and / or treat acute radiation pneumonitis and radiation pulmonary fibrosis, and the prevention and / or treatment effect is significantly better than that of single administration of amifostine, amifostine or polydatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The cytotoxicity of amlexanol and polydatin on RLE-6TN cell line;

[0020] Figure 2HE staining results of lung tissues of mice in each group on the 20th day after ionizing radiation;

[0021] Figure 3 This is the survival curve of mice with radiation lung injury model. DETAILED DESCRIPTION

[0022] The present invention provides the use of amlexanox combined with polydatin in the preparation of a medicine for preventing and / or treating radiation lung injury.

[0023] Amlexanox is a benzopyran-pyridinecarboxylic acid derivative that is clinically used to treat recurrent oral ulcers, asthma and allergic rhinitis.

[0024] Polydatin is a natural precursor of resveratrol extracted from the root of Polygonum cuspidatum. It has the effects of suppressing cough, regulating blood lipids, lowering cholesterol, and resisting shock.

[0025] In the present invention, the radiation lung injury is lung injury caused by ionizing radiation.

[0026] In the present invention, the radiation lung injury includes acute radiation pneumonitis or radiation pulmonary fibrosis.

[0027] In the present invention, the mass ratio of amlexanol to polydatin is 2-12:15-45, preferably 4-8:20-30, and more preferably 6:25.

[0028] The present invention also provides a pharmaceutical composition for preventing and / or treating radiation-induced lung injury, the pharmaceutical composition comprising amlexanox, polydatin and a pharmaceutically acceptable carrier.

[0029] In the present invention, the mass ratio of amlexanol to polydatin is 2-12:15-45, preferably 4-8:20-30, and more preferably 6:25.

[0030] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating radiation lung injury.

[0031] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating acute radiation pneumonitis.

[0032] The present invention also provides the use of the pharmaceutical composition in preparing a preparation for preventing and / or treating radiation pulmonary fibrosis.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Among the reagents of the present invention, amlexanox was purchased from Abcam, UK, polydatin was purchased from Sigma-Aldrich, USA, amifostine was purchased from Hubei Jianyuan Chemical Co., Ltd., CCK-8 reagent was purchased from Dojin Chemical Research Institute, Japan, and Tunel detection kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0035] In the test materials of the present invention, the test mice are 6-8 weeks old C57BL / 6 strain mice, weighing 18-22 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; the test cell line is rat type II alveolar epithelial cell RLE-6TN cell line (article number: Delf-16735), purchased from Hefei Wanwu Biotechnology Co., Ltd.

[0036] Example 1: Cytotoxicity of Amiloxanol and Polydatin

[0037] RLE-6TN cells were inoculated in DMFM / F12 medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO. 2 The cells were cultured under the conditions of 4% paraformaldehyde and 1% paraformaldehyde to the logarithmic growth phase, and then inoculated into a 96-well plate and divided into an amlexanox group, a polydatin group and a control group. After culturing for 24 hours, amlexanox was added to the culture wells of the amlexanox group to a final concentration of 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L and 200 μmol / L, respectively, and polydatin was added to the culture wells of the polydatin group to a final concentration of 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L and 200 μmol / L, respectively, and no reagent was added to the control group. After culturing for 48 hours, CCK-8 reagent was added to each culture well and cultured for 2 hours according to the instructions for use of CCK-8 reagent, and the OD of each culture well was measured by a microplate reader. 450nm The cell survival rate was calculated and the results were as follows Figure 1 shown.

[0038] The cell viability calculation formula is:

[0039]

[0040] The results showed that amlexanol at a final concentration of ≤50 μmol / L or polydatin at a final concentration of ≤100 μmol / L had no inhibitory effect on the growth and proliferation of the RLE-6TN cell line.

[0041] Example 2: The preventive and therapeutic effects of amlexanox combined with polydatin on radiation-induced lung injury

[0042] A total of 180 C57BL / 6 mice were divided into 9 groups, including blank control group, simple irradiation group, amlexanox group, polydatin group, low-dose combination group, medium-dose combination group, high-dose combination group, negative control group, and positive control group, with 20 mice in each group. One hour before ionizing radiation, the mice in the amlexanox group were injected intraperitoneally with 50 mg / kg of amlexanox, the mice in the polydatin group were injected intraperitoneally with 100 mg / kg of polydatin, the mice in the low-dose combination group were injected intraperitoneally with 2 mg / kg of amlexanox + 15 mg / kg of polydatin, the mice in the medium-dose combination group were injected intraperitoneally with 6 mg / kg of amlexanox + 25 mg / kg of polydatin, the mice in the high-dose combination group were injected intraperitoneally with 12 mg / kg of amlexanox + 45 mg / kg of polydatin, the mice in the blank control group were injected intraperitoneally with 0.1 mL of normal saline, the mice in the simple irradiation group and the negative control group were injected intraperitoneally with 0.1 mL of dimethyl sulfoxide, and the mice in the positive control group were injected intraperitoneally with 20 mg / kg of amifostine. The mice in the simple irradiation group, amlexanox group, polydatin group, low-dose combination group, medium-dose combination group, high-dose combination group, and positive control group were anesthetized by intraperitoneal injection of 50 mg / kg 1% sodium pentobarbital solution and then fixed in supine position. 60 Co was used as a radiation source to irradiate the lungs of each mouse with γ-ray ionizing radiation, with a radiation dose of 30 Gy and a radiation dose rate of 1 Gy / min. Lead plates were used to shield other parts of each mouse to avoid ionizing radiation damage. The mice in the blank control group and the negative control group were not irradiated. After the ionizing radiation, the bilateral lungs of the mice were removed on the 7th, 14th, and 20th days, fixed, wax-embedded, and sliced ​​for HE staining. The results are shown in Figure 2 As shown; the lungs of each mouse were washed with pre-cooled physiological saline, the fresh wet weight and the dry weight after drying were weighed, and the wet / dry specific gravity was calculated; the Tunel detection kit was used to determine the apoptosis rate of vascular endothelial cells in the lungs of each mouse. The specific steps refer to the instructions for use of the Tunel detection kit. The results are shown in Tables 1 to 2.

[0043] The formula for calculating wet / dry specific gravity is:

[0044] HE staining results showed that the lung tissue structure of mice in the blank control group was normal and there was no obvious pathological change, which was consistent with the negative control group. After 20 days of ionizing radiation, the lung tissue capillaries of mice in the simple irradiation group were dilated and congested, with obvious inflammatory reaction, local alveolar cavity collapse and atelectasis, severe interstitial edema, obvious widening and partial fusion of alveolar walls, and obvious hemorrhagic lesions and fibrinoid exudates in the alveolar cavity. The degree of lung tissue lesions of mice in the amlexanox group, polydatin group and positive control group was similar, with a certain degree of interstitial edema, widening and fusion of alveolar walls, but the overall degree of inflammation was milder than that in the simple irradiation group, and the alveolar structure and contour could still be found. Although the mice in the combination group still had pulmonary interstitial edema and varying degrees of alveolar wall fusion, the overall degree of inflammation was lower than that in the simple irradiation group, the amlexanox group and the polydatin group, and the degree of alveolar contour and structural lesions was also reduced; in the lung tissue of the mice in the medium-dose combination group, most of the alveolar cavity structures and contours were clear, the alveolar wall thickening was not obvious, the pulmonary interstitial edema was not serious, and there was no obvious fusion and adhesion between most of the alveolar walls. Although it could not completely reach the healthy level of the lung tissue structure of the blank control group and the negative control group mice, the degree of alveolar inflammation and the thickness of the alveolar wall were significantly lower than those in the amlexanox group, the polydatin group and the positive control group, and were close to the staining results of the mice in the high-dose combination group. The results showed that the administration of amlexanox, polydatin or amifostine alone could prevent, treat and alleviate radiation-induced lung injury to a certain extent, and there was no significant difference in the preventive and therapeutic effects on radiation-induced lung injury; the combination of amlexanox and polydatin had a certain effect on the prevention and treatment of radiation-induced lung injury at low doses, which preliminarily reflected the synergistic effects of anti-radiation and anti-inflammatory effects; it could show significant preventive and therapeutic effects at medium and high doses, and was not dose-dependent.

[0045] The wet / dry weight ratio of mouse lungs can be used to indicate the bleeding of lung tissue, thus indirectly indicating the degree of vascular damage; the Tunel detection kit can be used to indicate the apoptosis of mouse vascular endothelial cells. Apoptotic cells can be dyed brown. By comparing the proportion of brown cells (i.e., the apoptosis rate of vascular endothelial cells), the degree of apoptosis of mouse vascular endothelial cells can be reflected, thereby indicating the damage of ionizing radiation to mouse lung blood vessels. The results showed that the wet / dry weight ratio of the lungs of mice in the simple irradiation group increased compared with the blank control group, and the apoptosis rate of vascular endothelial cells increased significantly, indicating that ionizing radiation can cause serious damage to the lung blood vessels and other tissues of mice, resulting in increased lung bleeding; the wet / dry weight ratio of the lungs and the apoptosis rate of vascular endothelial cells of mice in the amlexanox group, polydatin group, low-dose combination group, medium-dose combination group, high-dose combination group and positive control group were all reduced, and the wet / dry weight ratio of the lungs of mice in the medium-dose combination group and high-dose combination group was significantly increased. The reduction in the apoptosis rate of vascular endothelial cells was more significant; this shows that the administration of amlexanox or polydatin alone can prevent and treat radiation-induced lung injury caused by ionizing radiation to a certain extent, and the preventive and therapeutic effect is similar to that of amifostine; while the combination of amlexanox and polydatin has a better preventive and therapeutic effect on radiation-induced lung injury caused by ionizing radiation, and the preventive and therapeutic effects of 6mg / kg amlexanox + 25mg / kg polydatin and 12mg / kg amlexanox + 45mg / kg polydatin on radiation-induced lung injury are similar.

[0046] Table 1 Wet / dry weight of lungs of mice in each group

[0047] Group No. 7d 14d 20th day Blank control group 0.45 0.42 0.44 Irradiation group 0.58 0.64 0.56 Amlexanox 0.49 0.51 0.51 Polydatin Group 0.53 0.56 0.54 Low-dose combination group 0.48 0.47 0.47 Medium dose combination group 0.45 0.44 0.43 High-dose combination group 0.46 0.43 0.43 Negative control group 0.44 0.42 0.44 Positive control group 0.48 0.50 0.50

[0048] Table 2 Apoptosis rate of vascular endothelial cells in each group of mice

[0049]

[0050]

[0051] Example 3: Effects of Amlexanox combined with Polydatin on TNF-α and IL-6

[0052] The lung tissue and blood samples of each group of mice described in Example 2 were taken, and the expression levels of TNF-α in the lung tissue of each mouse and the expression levels of IL-6 in the blood samples were measured by Western blot and ELISA, respectively, according to the literature published by Bao Pengtao et al. (Bao Pengtao, Qi Haowen, Gao Wei, et al. Effects of ulinastatin on TNF-α and IL-6 in radiation-induced lung injury in rats [J]. Chinese Journal of Radiological Medicine and Protection, 2009, 29(2): 154-157). The results are shown in Table 3.

[0053] TNF-α is the initiator of the cytokine regulatory network, which can induce the production of inflammatory mediators, promote fibrosis and stimulate the release of cytokines such as IL-6, thereby producing a cytokine cascade effect; IL-6 can be used as an indicator to predict the severity of radiation-induced lung injury. The results showed that the expression of TNF-α and IL-6 in the mice in the simple irradiation group was significantly higher than that in the other groups; the expression of TNF-α and IL-6 in the mice in the amlexanox group, polydatin group and positive control group was significantly lower than that in the simple irradiation group, and there was no significant difference with the low-dose combination group; while the expression of TNF-α and IL-6 in the mice in the medium-dose combination group and the high-dose combination group was significantly different from that in the positive control group. This indicates that the administration of amlexanox or polydatin alone can inhibit the production of IL-6 to a certain extent, and the inhibitory effect is equivalent to that of amifostine; and the combination of amlexanox and polydatin can more effectively inhibit the expression of TNF-α and the production and release of IL-6, thereby effectively preventing and treating radiation-induced lung injury.

[0054] Table 3 Expression of TNF-α and IL-6 in each group of mice

[0055]

[0056]

[0057] Example 4: Establishment of a mouse model of radiation-induced lung injury

[0058] C57BL / 6 mice were anesthetized by intraperitoneal injection of 50 mg / kg 1% sodium pentobarbital solution and fixed in supine position. 60 Co was used as a radiation source to perform local γ-ray ionizing radiation on the chest of C57BL / 6 mice, with a radiation dose of 30Gy and a radiation dose rate of 200cGy / min, and lead plates were used to shield other parts of the C57BL / 6 mice to avoid ionizing radiation damage. After ionizing radiation, C57BL / 6 mice showed acute radiation pneumonitis within 1 to 2 weeks, and fibrosis symptoms appeared in bilateral lung tissues within 3 to 5 weeks, which was basically consistent with the course of radiation lung injury in humans, indicating that the radiation lung injury model mouse model was successfully established.

[0059] Example 5: Effect of Amlexanox combined with Polydatin on the survival rate of mice with radiation-induced lung injury model

[0060] 75 mice with radiation lung injury model constructed in Example 3 were selected and divided into 5 groups, namely, irradiation group, amlexanox group, polydatin group, combination group, and positive control group, with 15 mice in each group. 6 hours after ionizing radiation, the mice in the amlexanox group were intraperitoneally injected with 50 mg / kg of amlexanox, the mice in the polydatin group were intraperitoneally injected with 100 mg / kg of polydatin, the mice in the combination group were intraperitoneally injected with 6 mg / kg of amlexanox + 25 mg / kg of polydatin, and the mice in the positive control group were intraperitoneally injected with 20 mg / kg of amifostine. Thereafter, the corresponding drugs were administered once every 2 days, and the mice in the irradiation group were not administered. The survival rate of mice in each group within 20 days was counted, and the results are as follows: Figure 3 shown.

[0061] The results showed that mice in the irradiation group began to die on the 5th day after ionizing radiation, and the survival rate on the 20th day was only 13.33%; mice in the amlexanox group and the positive control group died on the 8th to 13th day, and the survival rate of mice in the amlexanox group was higher than that in the positive control group before the 14th day, with a survival rate of 33.33% on the 20th day; the death time of mice in the polydatin group continued until the 15th day; only a small number of mice in the combination group died on the 8th to 12th day, and the survival rate on the 20th day was 86.67%. The results showed that the combination of amifostine and polydatin can significantly improve the survival rate of mice with radiation lung injury. Compared with the administration of amifostine or amifostine alone, the combination of amifostine and polydatin increased the survival rate of mice with radiation lung injury model by 53.34%. Compared with the administration of amifostine or polydatin alone, the combination of amifostine and polydatin significantly reduced the dosage of amifostine and polydatin.

[0062] As can be seen from the above examples, the present invention provides the use of amlexanox combined with polydatin in the preparation of a drug for preventing and / or treating radiation lung injury. Compared with the prior art, the present invention combines amlexanox with polydatin, which can significantly reduce the dosage of amlexanox and polydatin, and improve the survival rate of radiation lung injury model mice by 53.34%, more effectively inhibit the expression of TNF-α and the production and release of IL-6, and will not produce any toxic side effects on normal alveolar epithelial cells, and has the advantages of small dosage, excellent prevention and / or treatment effect, no toxic side effects, no dose dependence, etc.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of amiloride combined with polydatin in the preparation of drugs for preventing and / or treating radiation-induced lung injury.

2. The use according to claim 1, characterized in that: The radiation lung injury is lung injury caused by ionizing radiation.

3. The use according to claim 1, characterized in that: The radiation lung injury includes acute radiation pneumonitis or radiation pulmonary fibrosis.

4. The use according to any one of claims 1 to 3, characterized in that: The mass ratio of amlexanol to polydatin is 2-12:15-45.

5. A pharmaceutical composition for preventing and / or treating radiation-induced lung injury, characterized in that: The pharmaceutical composition comprises amlexanox, polydatin and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The mass ratio of amlexanol to polydatin is 2-12:15-45.

7. Use of the pharmaceutical composition according to claim 5 or 6 in the preparation of a preparation for preventing and / or treating radiation lung injury.

8. Use of the pharmaceutical composition according to claim 5 or 6 in the preparation of a preparation for preventing and / or treating acute radiation pneumonitis.

9. Use of the pharmaceutical composition according to claim 5 or 6 in the preparation of a preparation for preventing and / or treating radiation-induced pulmonary fibrosis.

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