Use of spermidine in the preparation of a medicament for preventing or treating radiation-induced xerostomia

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to the use of spermidine in the preparation of a drug for preventing or treating radiation-induced xerostomia. Aiming at the problems of poor efficacy and many side effects of existing drugs for treating radiation-induced xerostomia, the present invention provides the use of spermidine in the preparation of a drug for preventing or treating radiation-induced xerostomia. The radiation-induced xerostomia occurs in head and neck tumors. The administration method of the spermidine is to feed water 7-10 days before irradiation, and the dosage used is 3 mM-5 mM. The present invention proves that spermidine has a certain therapeutic effect on the xerostomia that occurs in C57BL / 6 mice after irradiation, and can restore the saliva flow rate and the number of aquaporin 5 in the mice, providing a promising treatment method for radiation-induced xerostomia.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of spermidine in the preparation of a drug for preventing or treating radiation-induced xerostomia. Background Art

[0002] Radiotherapy is an important treatment modality for head and neck malignancies. With the development of radiotherapy equipment and technology, the exposure of normal tissues has been minimized to the greatest extent, and the acute and late reactions have decreased compared with conventional radiotherapy. However, due to the inherent nature of radiation and the anatomical location of the primary focus of head and neck tumors involving or adjacent to the oral cavity, radiotherapy still delivers low to moderate doses of radiation to the oral cavity. Therefore, patients undergoing head and neck radiotherapy may develop various oral complications during or after radiotherapy, including: xerostomia, oral mucositis, halitosis, dental caries, and taste disorders, etc. According to previous reports, the incidence of xerostomia in patients with head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) receiving radiotherapy or concurrent chemoradiotherapy is above 90%, and the incidence of grade 3 or above xerostomia exceeds 50%. In mild cases, it is manifested as local discomfort or pain in the oral cavity or throat, and obvious taste abnormalities may also occur. In severe cases, dysphagia occurs, resulting in the inability to eat, and even forced interruption of treatment. On the one hand, it affects the curative effect of tumor treatment, which may affect the long-term survival of patients. On the other hand, it also seriously affects the quality of life of patients.

[0003] The main cause of radiation-induced xerostomia is damage to the submandibular gland, which affects saliva secretion. Currently, drugs such as artificial saliva and oral saliva stimulants are often used for treatment, but the effect is not ideal, and there is an urgent need to develop new treatment methods to restore the saliva function of patients.

[0004] As one of the polyamine members, spermidine is a trivalent cationic compound found in eukaryotic cells. It can interact with polyanions such as nucleic acids, proteins, and ATP through electrostatic binding, further maintaining DNA genome homeostasis, regulating gene transcription and translation, regulating autophagy, apoptosis, oxidative stress, angiogenesis, and cell communication, and is indispensable in cell division and proliferation. A number of studies have shown that spermidine has functions significantly different from other biogenic amines. In human epidemiological studies, the intake of polyamines in the diet is associated with a reduction in cardiovascular and cancer-related mortality. The results of animal-related studies show that the natural polyamine spermidine has significant cardioprotective and neuroprotective effects and stimulates anti-cancer immune surveillance in rodent models. In addition, spermidine can also enhance mitochondrial metabolism, exert anti-inflammatory properties, and prevent stem cell senescence.

[0005] However, at present, there is no report on whether spermidine has an impact on radiation-induced xerostomia. Summary of the Invention

[0006] The technical problem to be solved by the present invention is: the existing drugs for treating radiation-induced xerostomia have poor curative effects and many side effects, etc.

[0007] The technical solution of the present invention to solve the above technical problems is: to provide a use of spermidine in the preparation of a drug for preventing or treating radiation-induced xerostomia.

[0008] Among them, in the above use, the radiation-induced xerostomia refers to the xerostomia symptoms caused by radiotherapy. In the present invention, a decrease in salivary flow rate is considered to have xerostomia symptoms.

[0009] Among them, in the above use, the radiation-induced xerostomia occurs in head and neck tumors.

[0010] Among them, in the above use, the administration method of spermidine is oral administration before irradiation.

[0011] Among them, in the above use, the dosage of spermidine is 3-10 mM; preferably 3 mM.

[0012] A drug for preventing or treating radiation-induced xerostomia, which contains spermidine.

[0013] Preferably, in the above drug, the dosage of spermidine in the drug is 3-10 mM; preferably 3 mM.

[0014] Preferably, in the above drug, it further includes pharmaceutically acceptable excipients.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The present invention first constructs an animal model of radiation-induced xerostomia, and through this model, it is found that spermidine can play a role in alleviating xerostomia caused by radiotherapy, can improve the xerostomia symptoms of mice, and can be prepared into a drug for preventing or treating radiation-induced xerostomia. The present invention also studies the administration route and dosage of spermidine and obtains a relatively effective treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The following shows a schematic diagram of radiotherapy: Among them, A represents the shielding and exposure positions of the radiotherapy lead block; B represents the body position of the mouse.

[0018] Figure 2 The following shows a statistical chart of the comparison of the curative effects of different administration routes and dosages.

[0019] Figure 3 The following shows a statistical chart of the comparison of oral moisture between the simple irradiation group and the spermidine irradiation treatment group.

[0020] Figure 4 The following shows a schematic diagram of mouse treatment: Water is fed to the mice 7 days before spermidine irradiation, and the salivary flow rate is measured on the 4th and 8th days after irradiation.

[0021] Figure 5The figure shows the verification of the therapeutic effect by staining the submandibular glands of mice in the prevention group and the simple irradiation group: Among them, A represents HE staining; B represents AQP5 immunohistochemical staining; C represents TUNEL immunofluorescence staining.

[0022] Figure 6 The figure shows the statistical chart of the therapeutic effect comparison between the prevention group and the simple irradiation group of mice: Among them, A represents the statistical chart of oral moisture measurement; B represents the immunohistochemical statistical chart; C represents the TUNEL immunofluorescence statistical chart. Specific implementation manners

[0023] The present invention has for the first time established a unique animal dry mouth model. By using the method of covering part of the tissue with lead blocks to expose it, the human dry mouth symptoms have been successfully replicated in mice. This is a unique and innovative method. Different from the past, our model more precisely simulates the human dry mouth symptoms, thus providing a more accurate test platform. The model of the present invention has (1) higher simulation accuracy: The new model more accurately simulates the pathophysiological process of human dry mouth: HE staining shows that radiation causes degeneration, edema of acinar cells, pyknosis and apoptotic necrosis of cell nuclei; atrophy of glandular parenchyma and interstitial fibrosis; dilation of ducts (intercalated ducts and striated ducts). (2) More reproducible results: The new model reduces the variability between results. (3) Optimized research time: A single 15Gy irradiation can shorten the modeling time compared with multiple small doses. (4) Reducing the pain of animals: Sacrificing animals within one week after irradiation reduces the pain of animals.

[0024] After the establishment of the dry mouth model of the present invention, it was screened that spermidine can significantly improve the dry mouth symptoms of mice. Different administration routes and doses were studied, including direct water feeding with spermidine concentration gradients of 10 mM, 5 mM and 3 mM; intraperitoneal injection of 50 mg / kg, 14 mg / kg, 3.6 mg / kg. Finally, it was found that when 3 mM was fed by water, it could effectively relieve the radiation-induced dry mouth of mice.

[0025] By establishing a mouse dry mouth model and using spermidine for treatment, the present invention can not only promote the basic research on dry mouth, but also contribute to the development of new drugs and clinical trials. The wide applicability and great potential of this method give our technology significant advantages over the existing technologies.

[0026] The following will further explain the specific implementation manners of the present invention through examples, but it does not mean that the protection scope of the present invention is limited to the scope described in the examples.

[0027] The mice described in the examples were purchased from Jicui Yakang Company; the rest of the reagents are all ordinary commercially available products.

[0028] Example 1 Construction of a mouse model of radiation-induced dry mouth

[0029] The specific experimental operations are as follows:

[0030] Six- to eight-week-old C57BL / 6 mice were purchased, weighed, and mice weighing about 20 g were selected.

[0031] The mice were randomly assigned to a simple radiation group and a spermidine prevention group, with 6 mice in each group. The two groups of mice were anesthetized with 1% sodium pentobarbital and fixed supine in a lead block box. The upper lead block baffle was adjusted to expose the submandibular gland area. Both groups of mice were given a dose of 15 Gy: 2.6 Gy / min (160 KV, 25 mA, 0.3 mm copper alloy filter) in one dose. The specific situation is as Figure 1 shown. When the saliva flow rate of the irradiated mice decreased, it was determined that the mice had developed radioactive xerostomia symptoms.

[0032] Example 2 Screening of the administration route and dose of spermidine

[0033] To screen different administration routes and doses of spermidine, we used two methods: directly adding water with spermidine concentration gradients of 10 mM, 5 mM, and 3 mM; and intraperitoneal injection with spermidine concentration gradients of 50 mg / kg, 14 mg / kg, and 3.6 mg / kg to find the best treatment method.

[0034] The results are as Figure 2 shown. The results showed that in the group given water directly, the best effect was achieved at a spermidine concentration gradient of 3 mM; in the intraperitoneal injection group, both spermidine concentration gradients of 14 mg / kg and 3.6 mg / kg were effective, but the best therapeutic effect was achieved with direct water administration of 3 mM and intraperitoneal injection of 14 mg / kg. Given that intraperitoneal injection cannot be applied to patients clinically, we chose to directly administer 3 mM of spermidine in water for the subsequent experiments.

[0035] At the same time, we also studied the effect of directly administering 3 mM of spermidine in water for 7 days after radiation. The results are as Figure 3 shown, and the therapeutic effect was not as good as that of pre-radiation administration.

[0036] Example 3 Study on the effect of spermidine on mice with radioactive xerostomia

[0037] Before irradiating the mice, the mice in the spermidine prevention group were given water containing 3 mM of spermidine, and the simple radiation group was only given basic sterilized water. The specific situation is as Figure 4 shown.

[0038] (1) Oral moisture measurement was performed on the mice in the spermidine prevention group and the simple radiation group. The specific operation method was as follows: Saliva was collected at the same time every morning. The mice were fasted for two hours before collection. Pilocarpine (0.5 mg / ml) was intraperitoneally injected at a dose of 25 μl per mouse. Saliva collection started within 2 minutes after pilocarpine administration. Cotton was placed in a 1.5 ml EP tube, weighed dry, then stuffed into the mouse's mouth. After continuously collecting saliva for 10 minutes, the cotton was taken out and put back into the 1.5 ml EP tube to weigh the wet weight. The calculation method of the collected saliva flow rate SFR (μL / min) was (wet weight - dry weight) (mg) / collection time (min) (assuming the specific gravity of saliva is 1 mg / mL).

[0039] The results showed that the average weight of oral saliva in the simple irradiation group of mice was 16 mg / min, and the average oral saliva in the spermidine prevention group of mice was 22 mg / min. This result was statistically significant (P < 0.05). The specific situation was as Figure 5 . It can be seen that oral administration of spermidine in advance can prevent the occurrence of radioactive xerostomia symptoms.

[0040] (2) Histological, immunohistochemical and immunofluorescence evaluations

[0041] On the day of irradiation, after measuring the saliva flow rate of the mice, the mice were sacrificed, and the submandibular gland tissues were taken for fixation with paraformaldehyde, flushing, dehydration, clearing, impregnation with wax, embedding, sectioning, and then staining evaluation.

[0042] The experimental results were as Figure 6 shown.

[0043] HE staining showed that the spermidine prevention group could significantly reduce acinar vacuolization, indicating that prophylactic administration of spermidine could play a role in protecting submandibular gland acinar cells.

[0044] Aquaporin 5 staining showed that the positive area in the prevention group increased by 5%. This result was statistically significant (P < 0.05); indicating that prophylactic administration of spermidine could play a role in protecting the secretory function of the submandibular gland.

[0045] TUNLE staining showed that the number of apoptotic cells per field decreased by 6. This result was statistically significant (P < 0.001). It indicated that prophylactic administration of spermidine could play a role in reducing apoptosis and protecting submandibular gland cells.

Claims

1. Use of spermidine as the sole active ingredient in the preparation of a drug for preventing or treating radiation-induced xerostomia, wherein the radiation-induced xerostomia refers to the dry mouth symptoms caused by radiotherapy, the radiation-induced xerostomia occurs in head and neck tumors, the administration method of the spermidine is oral administration before irradiation, and the dosage of the spermidine is 3-10 mM.

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