Application of amlodipine combined with polydatin in preparation of preparation for preventing and / or treating radiation-induced lung injury

Abstract

The application relates to application of aminolevulinic acid and rhizomaleae in preparation of preparations for preventing and / or treating radiation-induced lung injury, and belongs to the technical field of medicines. The application provides application of aminolevulinic acid and rhizomaleae in preparation of medicines for preventing and / or treating radiation-induced lung injury. The aminolevulinic acid and the rhizomaleae are combined to prevent and / or treat radiation-induced lung injury caused by ionizing radiation, and under the premise of significantly reducing the administration dose of the aminolevulinic acid and the rhizomaleae, the survival rate of a radiation-induced lung injury model mouse is increased by more than 50%, and the application has the characteristics of small drug dosage, excellent prevention and / or treatment effect, no toxic side effect and no dose dependence.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of amlexanox combined with hovenine in the preparation of preparations for preventing and / or treating radiation-induced lung injury. BACKGROUND

[0002] Radiotherapy is one of the clinical treatment methods for chest malignant tumors. During radiotherapy, lung tissue will inevitably be exposed to a certain dose of ionizing radiation, thereby causing different degrees of radiation-induced lung injury (RILI). RILI includes acute radiation pneumonitis (RP) and radiation-induced pulmonary fibrosis (RPF), wherein RP, as an early manifestation, often occurs within 1-3 months after the start of radiotherapy, and RPF often occurs as a late event. The main clinical manifestations of RILI are inflammatory infiltration of alveolar interstitium, progressive dyspnea, and deterioration of lung function, which can even lead to respiratory failure, 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 application at present. However, due to its obvious side effects such as causing hypotension, severe nausea, and poor tolerance, its clinical application is limited. 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 adverse symptoms such as vomiting, diarrhea, dizziness, and headache. Therefore, there is no drug in the prior art that can stably and effectively prevent and / or treat radiation-induced lung injury with a small amount, high efficiency, low or no toxicity. SUMMARY

[0004] The present application aims to provide the application of amlexanox combined with hovenine in the preparation of preparations for preventing and / or treating radiation-induced lung injury, so as to solve the problems of large dosage, many side effects, poor prevention effect, and poor 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 application purposes, the present application provides the following technical solutions:

[0006] The present application provides the application of amlexanox combined with hovenine in the preparation of a drug for preventing and / or treating radiation-induced lung injury.

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

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

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

[0010] The present application also provides a pharmaceutical composition for preventing and / or treating radiation-induced lung injury, which comprises amifostine, polydatin and a pharmaceutically acceptable carrier.

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

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

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

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

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

[0016] The present application can effectively prevent and / or treat radiation-induced lung injury caused by ionizing radiation by using amifostine and polydatin in combination, and the dosage of amifostine and polydatin in combination is significantly lower than that of amifostine or polydatin alone.

[0017] The present application can effectively treat radiation-induced lung injury caused by ionizing radiation by using amifostine and polydatin in combination, and amifostine and polydatin in combination can increase the survival rate of radiation-induced lung injury model mice by more than 50% under the premise of significantly reducing the dosage of amifostine and polydatin, more effectively inhibit the expression of TNF-α and the production and release of IL-6, and at the same time, do not produce any toxic side effects on normal alveolar epithelial cells, with the advantages of small dosage, excellent treatment effect, no toxic side effects and no dose dependence.

[0018] The present application can prevent and / or treat acute radiation pneumonitis and radiation-induced pulmonary fibrosis, and the prevention and / or treatment effect is significantly better than that of amifostine, amifostine or polydatin alone. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Cytotoxicity of amifostine and polydatin to 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 Survival curves of the model mice of radiation-induced lung injury. DETAILED DESCRIPTION

[0022] The present application provides the use of Amlexanox and Polydatin in the preparation of a medicament for preventing and / or treating radiation-induced lung injury.

[0023] Amlexanox is a benzopyran-pyridine carboxylic acid derivative, which is clinically used for treating recurrent oral ulcer, asthma and allergic rhinitis.

[0024] Polydatin is a natural precursor of resveratrol extracted from the roots of Polygonum cuspidatum, which has the effects of relieving cough, regulating blood lipids, reducing cholesterol and resisting shock.

[0025] In the present application, the radiation-induced lung injury is lung injury caused by ionizing radiation.

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

[0027] In the present application, the mass ratio of Amlexanox to Polydatin is 2-12:15-45, preferably 4-8:20-30, and further preferably 6:25.

[0028] The present application also provides a pharmaceutical composition for preventing and / or treating radiation-induced lung injury, which comprises Amlexanox, Polydatin and a pharmaceutically acceptable carrier.

[0029] In the present application, the mass ratio of Amlexanox to Polydatin is 2-12:15-45, preferably 4-8:20-30, and further preferably 6:25.

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

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

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

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

[0034] The amlexanox is purchased from Abeam Company in the UK, the polydatin is purchased from Sigma-Aldrich Company in the United States, the amifostine is purchased from Hubei Jianyuan Chemical Co., Ltd., the CCK-8 reagent is purchased from TCI Chemical Industry Co., Ltd., and the Tunel detection kit is purchased from Yixing Biotechnology Co., Ltd.

[0035] In the test material of the present application, the test mice are 6-8-week-old C57BL / 6 strain mice with a body weight of 18-22 g, which are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; and the test cell line is a rat type II alveolar epithelial cell RLE-6TN cell line (item number: Delf-16735), which is purchased from Hefei All Things Biological Technology Co., Ltd.

[0036] Example 1: Cytotoxicity of amlexanox and polydatin

[0037] The RLE-6TN cell line is inoculated in DMFM / F12 culture solution containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 until the logarithmic growth phase, and then inoculated in a 96-well plate and divided into an amlexanox group, a polydatin group and a control group. After 24 h of continuous culture, amlexanox is 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, polydatin is 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 is added in the control group; after 48 h of continuous culture, CCK-8 reagent is added to each culture well according to the instructions of the CCK-8 reagent and cultured for 2 h, and the OD value of each culture well is determined by an enzyme marker and the cell survival rate is calculated, and the results are shown in 450nm Figure 1

[0038] The cell survival rate calculation formula is:

[0039]

[0040] The results show that amlexanox with a final concentration of ≤50 μmol / L or polydatin with a final concentration of ≤100 μmol / L does not inhibit the growth and proliferation of the RLE-6TN cell line.

[0041] Example 2: Prevention and treatment effect of amlexanox combined with polydatin on radiation-induced lung injury

[0042] ​​180 C57BL / 6 mice were divided into 9 groups, namely, blank control group, simple irradiation group, amlexanox group, astilbin 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 intraperitoneally injected with 50 mg / kg amlexanox, the mice in the astilbin group were intraperitoneally injected with 100 mg / kg astilbin, the mice in the low-dose combination group were intraperitoneally injected with 2 mg / kg amlexanox + 15 mg / kg astilbin, the mice in the medium-dose combination group were intraperitoneally injected with 6 mg / kg amlexanox + 25 mg / kg astilbin, the mice in the high-dose combination group were intraperitoneally injected with 12 mg / kg amlexanox + 45 mg / kg astilbin, the mice in the blank control group were intraperitoneally injected with 0.1 mL normal saline, the mice in the simple irradiation group and the negative control group were intraperitoneally injected with 0.1 mL dimethyl sulfoxide, and the mice in the positive control group were intraperitoneally injected with 20 mg / kg amifostine. After the mice in the simple irradiation group, the amlexanox group, the astilbin group, the low-dose combination group, the medium-dose combination group, the high-dose combination group, and the positive control group were anesthetized by intraperitoneal injection of 50 mg / kg 1% sodium pentobarbital solution, they were fixed in a supine position, and the lung tissue was exposed. 60 The lung of each mouse was subjected to γ-ray ionizing radiation using Co as a radiation source, with a radiation dose of 30 Gy and a radiation dose rate of 1 Gy / min. Other parts of each mouse were shielded using a lead plate to avoid ionizing radiation damage. The mice in the blank control group and the negative control group were not subjected to ionizing radiation. After ionizing radiation, the bilateral lungs of the mice were removed on the 7th day, the 14th day, and the 20th day, and were fixed, embedded in wax blocks, sectioned, and subjected to HE staining. The results are shown in Table 1-2. Figure 2 The lung of each mouse was washed with pre-cooled normal saline, and the fresh wet weight and the dry weight after drying were measured and the wet / dry ratio was calculated. The vascular endothelial cell apoptosis rate of the lung of each mouse was determined using a Tunel detection kit, according to the instructions of the Tunel detection kit. The results are shown in Table 1-2.

[0043] The calculation formula of the wet / dry ratio is as follows:

[0044] HE staining results showed that the lung tissue structure of the blank control group mice was normal and had no obvious pathological changes, which was consistent with the negative control group; after 20 days of ionizing radiation, the lung tissue of the simple irradiation group mice had capillary dilation, hyperemia, obvious inflammatory reaction, local alveolar cavity collapse, interstitial edema, alveolar wall widening and partial fusion, and obvious hemorrhagic lesions and fibrin-like exudates in the alveolar cavity; the lung tissue of the aminoxifene group, polydatin group and positive control group mice had similar pathological changes, and there was a certain degree of interstitial edema, alveolar wall widening and fusion, but the overall inflammatory degree was lighter than that of the simple irradiation group, and the alveolar structure and outline could still be found; the lung interstitial edema and alveolar wall fusion of the low-dose combination group mice still existed, but the overall inflammatory degree was lower than that of the simple irradiation group, aminoxifene group and polydatin group, and the alveolar outline and structure pathological changes were also reduced; in the lung tissue of the medium-dose combination group mice, most of the alveolar cavity structure and outline were clear, the alveolar wall thickening was not obvious, the lung interstitial edema was not serious, and there was no obvious fusion and adhesion phenomenon 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 negative control group mice, but the alveolar inflammation degree and alveolar wall thickness were significantly lower than those of the aminoxifene group, polydatin group and positive control group, and were close to the staining results of the high-dose combination group mice. It was indicated that aminoxifene, polydatin or amifostine alone could prevent and relieve the radiation-induced lung injury to a certain extent, and there was no obvious difference in the prevention effect on radiation-induced lung injury; aminoxifene combined with polydatin had a certain effect on the prevention of radiation-induced lung injury at low doses, which initially embodied the synergistic effect of anti-radiation and anti-inflammatory; at medium and high doses, it could show significant prevention effect, and had no dose dependence.

[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 stained 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, the polydatin group, the low-dose combination group, the medium-dose combination group, the high-dose combination group and the positive control group were all reduced, and the wet / dry weight ratio of the lungs of mice in the medium-dose combination group and the high-dose combination group were 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 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, 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 7th day 14th day 20th day Blank control group 0.45 0.42 0.44 Simple irradiation group 0.58 0.64 0.56 Amlexanox group 0.49 0.51 0.51 Polystachoside 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] Lung tissue and blood samples from each group of mice described in Example 2 were taken, and the expression levels of TNF-α in the lung tissue and IL-6 in the blood samples of each mouse were measured by Western blot and enzyme-linked immunosorbent assay (ELISA) respectively, referring 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 a cytokine regulatory network initiator that can induce the production of inflammatory mediators, promote fibrosis, and stimulate the release of cytokines such as IL-6, thereby generating a cytokine cascade effect. IL-6 can be used as a predictor of the severity of radiation-induced lung injury. Results showed that the expression of TNF-α and IL-6 in mice in the irradiation-only group was significantly elevated compared with the other groups. However, the expression of TNF-α and IL-6 in mice in the amlexanox, polydatin, and positive control groups was significantly decreased compared with the irradiation-only group and did not differ significantly from the low-dose combination group. However, the expression of TNF-α and IL-6 in mice in the medium-dose combination and high-dose combination groups was significantly different from that in the positive control group. This suggests that either amlexanox or polydatin alone can inhibit IL-6 production to a certain extent, with an inhibitory effect comparable to that of amifostine. The combination of amlexanox and polydatin is more effective in inhibiting TNF-α expression and IL-6 production and release, thereby effectively preventing and treating radiation-induced lung injury.

[0054] Table 3 TNF-α and IL-6 expression levels in mice in each group

[0055]

[0056]

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

[0058] C57BL / 6 mice 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 locally irradiate the chest of C57BL / 6 mice with γ-rays at a dose of 30 Gy at a dose rate of 200 cGy / min. Lead plates were used to shield the rest of the mice from ionizing radiation damage. Following ionizing radiation, the C57BL / 6 mice developed acute radiation pneumonitis within 1 to 2 weeks and bilateral lung fibrosis within 3 to 5 weeks, a course consistent with human radiation lung injury, indicating that the mouse model of radiation lung injury 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] Select 75 mice of the radiation-induced lung injury model constructed in Example 3 and divide them into 5 groups, i.e. the irradiation group, the aminoxime group, the polydatin group, the combination group and the positive control group, 15 mice in each group. At 6 hours after ionizing radiation, the mice in the aminoxime group are injected intraperitoneally with 50 mg / kg aminoxime, the mice in the polydatin group are injected intraperitoneally with 100 mg / kg polydatin, the mice in the combination group are injected intraperitoneally with 6 mg / kg aminoxime + 25 mg / kg polydatin, and the mice in the positive control group are injected intraperitoneally with 20 mg / kg amifostine. Then, the corresponding drugs are administered once every 2 days. The mice in the irradiation group are not administered with any drug. The survival rates of the mice in each group within 20 days are counted, and the results are shown in Table 1. Figure 3

[0061] The results show that the mice in the irradiation group start to die at the 5th day after ionizing radiation, and the survival rate at the 20th day is only 13.33%. The mice in the aminoxime group and the positive control group start to die at the 8th-13th day, and the survival rate of the mice in the aminoxime group is higher than that of the mice in the positive control group before the 14th day. The survival rates of the mice in the aminoxime group and the positive control group at the 20th day are both 33.33%. The mice in the polydatin group start to die until the 15th day. The mice in the combination group only start to die at the 8th-12th day, and the survival rate at the 20th day is 86.67%. It is indicated that the combination of aminoxime and polydatin can significantly improve the survival rate of the mice with radiation-induced lung injury. Compared with the administration of amifostine or aminoxime alone, the combination of aminoxime and polydatin can improve the survival rate of the mice with radiation-induced lung injury by 53.34%. Compared with the administration of aminoxime or polydatin alone, the combination of aminoxime and polydatin can significantly reduce the administration dose of aminoxime and polydatin.

[0062] From the above examples, it is known that the present application provides the use of aminoxime combined with polydatin in the preparation of a drug for preventing and / or treating radiation-induced lung injury. Compared with the prior art, the combination of aminoxime and polydatin can improve the survival rate of the mice with radiation-induced lung injury by 53.34% under the premise of significantly reducing the administration dose of aminoxime and polydatin, more effectively inhibits the expression of TNF-α and the production and release of IL-6, and does not cause any toxic side effects to normal alveolar epithelial cells, and has the advantages of small administration dose, excellent prevention and / or treatment effect, no toxic side effects, no dose dependence, etc.

[0063] The above description is only the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. Use of amlexanox 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-induced lung injury is lung injury caused by ionizing radiation.

3. The use according to claim 1, characterized in that The radiation-induced lung injury includes acute radiation-induced pneumonitis or radiation-induced 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-induced 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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