阅读说明：本技术 叔丁基对苯二酚在制备预防和/或治疗粘膜炎的药物中的用途 (Application of tert-butyl hydroquinone in preparation of medicine for preventing and/or treating mucositis ) 是由 许颖 常晓松 吴东明 邓仕华 刘腾 于 2021-09-23 设计创作，主要内容包括：本发明提供了一种叔丁基对苯二酚在制备预防和/或治疗粘膜炎的药物中的用途,属于制药领域。本发明首次发现,叔丁基对苯二酚能够显著改善5-FU诱导的肠粘膜炎,能够改善腹泻,降低肠道长度损失,减轻病理变化(如粘膜损伤,坏死,组织结构丧失,水肿和炎症细胞浸润),降低炎症因子表达水平；本发明还发现,叔丁基对苯二酚对5-FU诱导的人肠上皮细胞损伤具有保护作用。因此,TBHQ在制备预防和/或治疗化疗导致的粘膜炎的药物、以及在制备对人肠上皮细胞损伤具有保护作用的药物中具有良好的应用前景。(The invention provides an application of tert-butyl hydroquinone in preparing a medicament for preventing and/or treating mucositis, belonging to the field of pharmacy. The invention discovers for the first time that tert-butyl hydroquinone can obviously improve 5-FU induced intestinal mucositis, can improve diarrhea, reduce intestinal length loss, relieve pathological changes (such as mucosal injury, necrosis, tissue structure loss, edema and inflammatory cell infiltration) and reduce the expression level of inflammatory factors; the invention also discovers that the tert-butyl hydroquinone has a protective effect on the 5-FU-induced damage of the human intestinal epithelial cells. Therefore, TBHQ has good application prospect in preparing medicaments for preventing and/or treating mucositis caused by chemotherapy and medicaments with protective effect on human intestinal epithelial cell injury.)

1. Use of tert-butylhydroquinone in the manufacture of a medicament for the prevention and/or treatment of mucositis.

2. Use according to claim 1, characterized in that: the mucositis is a gastrointestinal mucositis.

3. Use according to claim 2, characterized in that: the gastrointestinal mucositis is intestinal mucositis.

4. Use according to any one of claims 1 to 3, characterized in that: the mucositis is caused by a chemotherapeutic drug, preferably 5-FU.

5. Use according to claim 4, characterized in that: the medicine can improve diarrhea, reduce intestinal length loss, improve mucosa injury, and reduce inflammatory factor expression level.

6. The application of tert-butyl hydroquinone in preparing medicine with protective effect on human intestinal epithelial cell injury.

7. Use according to claim 6, characterized in that: the human intestinal epithelial cell injury is human intestinal epithelial cell injury caused by a chemotherapeutic drug, preferably 5-FU.

8. Use according to any one of claims 1 to 7, characterized in that: the medicine is a preparation prepared by taking tert-butyl hydroquinone as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

9. Use according to claim 8, characterized in that: the preparation is an oral preparation.

10. Use according to claim 8, characterized in that: the preparation is an injection preparation.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to application of tert-butyl hydroquinone in preparation of a medicament for preventing and/or treating mucositis.

Background

Chemotherapy is one of the main methods for treating malignant tumors, but researches show that the incidence rate of mucositis is up to 90% when chemotherapeutics such as Methotrexate (MTX), 5-fluorouracil (5-FU), cytarabine, vincristine and etoposide are applied. The occurrence of the chemotherapy mucositis not only causes the treatment cost of the patient to rise and the hospitalization time to be prolonged, but also increases the physical and psychological pain of the patient and reduces the life quality of the patient. Therefore, the development of the medicine for effectively treating the chemotherapeutic mucositis has important significance.

The incidence of the gastrointestinal mucositis caused by chemotherapy is 40%, the gastrointestinal mucositis caused by chemotherapy is not only limited to oral mucosa injury, such as oral mucosa erythema, desquamation, pharyngalgia and oral ulcer, but also comprises gastrointestinal mucosa injury, and the clinical table shows that: nausea, vomiting, loss of appetite, abdominal pain, abdominal distension, diarrhea, perianal abscess, etc. Bacterial infection caused by alimentary tract mucositis can cause serious systemic infection of patients, so that the usage rate of nasogastric tubes, parenteral nutrition and the number of times of venipuncture of the patients are increased, the dosage and the use frequency of analgesics are increased, the treatment compliance of the patients is reduced, the original chemotherapy scheme is changed or interrupted, and the anti-tumor treatment effect of the patients is influenced.

Intestinal mucositis caused by chemotherapy is a kind of alimentary tract mucositis which appears in the course of chemotherapy, has the characteristics of high incidence and great harm, and seriously affects the treatment and life quality of tumor patients. Because the intestinal mucosa epithelium is updated quickly and is sensitive to chemotherapy, after receiving gastrointestinal interventional chemotherapy, the intestinal mucosa epithelium is updated slowly or even stops, mucosal edema, abscission and ulcer appear, the submucosa and the lamina propria are naked, and the clinical manifestations are different degrees of digestive tract symptoms, such as nausea, vomiting, abdominal pain, diarrhea and digestive tract hemorrhage. At present, few drugs are reported for treating chemotherapy-induced intestinal mucositis.

Tert-butyl hydroquinone, TBHQ for short, and has the structureThe research finds that the tert-butyl hydroquinone can relieve high-sugar-induced oxidative stress injury of mesangial cells, and the action mechanism of the tert-butyl hydroquinone is probably realized by activating an Nrf2-ARE signal pathway and further enhancing the expression of antioxidant proteins, HO-1 and gamma-GCS. However, there has been no report of the use of t-butylhydroquinone in the treatment of chemotherapy-induced mucositis.

Disclosure of Invention

The invention aims to provide a new application of tert-butyl hydroquinone in preparing a medicament for preventing and/or treating mucositis. The invention also aims to provide a new application of the tert-butyl hydroquinone in preparing medicaments with protective effect on human intestinal epithelial cell injury.

The invention provides an application of tert-butyl hydroquinone in preparing a medicament for preventing and/or treating mucositis.

Further, the mucositis is a digestive tract mucositis.

Further, the gastrointestinal mucositis is intestinal mucositis.

Further, the mucositis is caused by a chemotherapeutic drug, preferably 5-FU.

Further, the medicine can improve diarrhea, reduce intestinal length loss, improve mucosal injury and reduce the expression level of inflammatory factors.

The invention also provides application of the tert-butyl hydroquinone in preparing a medicament with a protective effect on human intestinal epithelial cell injury.

Further, the human intestinal epithelial cell injury is a chemotherapy drug induced human intestinal epithelial cell injury, preferably the chemotherapy drug is 5-FU.

Furthermore, the medicine is a preparation prepared by taking tert-butyl hydroquinone as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Further, the formulation is an oral formulation.

Further, the preparation is an injection preparation.

Tert-butyl hydroquinone, abbreviated as TBHQ.

The invention discovers for the first time that TBHQ has a protective effect on 5-FU-induced human intestinal epithelial cell damage, and tert-butylhydroquinone can be used for preparing a medicine having a protective effect on human intestinal epithelial cell damage.

The invention discovers for the first time that TBHQ can obviously improve 5-FU induced intestinal mucositis, can improve diarrhea, reduce intestinal length loss, relieve pathological changes (such as mucosal injury, necrosis, tissue structure loss, edema and inflammatory cell infiltration) and reduce the expression level of inflammatory factors. Therefore, TBHQ has good application prospect in preparing medicines for preventing and/or treating intestinal mucositis caused by chemotherapy.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 results of experiments with TBHQ to reduce 5-FU-induced damage to human intestinal epithelial cells. Note: (A) CK-8 cell viability assays were performed 24 and 48 hours after treatment with 5-FU at various concentrations, with half the lethal dose occurring at approximately 48 hours after 5 μ M treatment. Subsequent experiments treated HIEC cells with 5 μ M5-FU and further co-cultured for 48h with TBHQ at different concentrations (5, 10 and 20 μ M), (B) LDH release was determined with CytoTox96 kit; (C) measuring the cell activity by using a CCK-8 detection kit; (D) a representative flow cytometer histogram; (E) representative 7-AAD stained fluorescence images. P <0.05, P <0.01, P < 0.001.

FIG. 2. results of experiments with TBHQ to reduce 5-FU-induced intestinal mucositis in mice. Note: (A) dosing and schedule of effect of TBHQ on 5-FU induced intestinal mucositis in mice. TBHQ (10mg/kg DMSO, i.p.) or vehicle (DMSO, i.p.) was administered from day 1 to day 8, and 5-FU (50mg/kg, i.p.) or vehicle (saline, i.p.) was administered from day 1 to day 5. Animals were sacrificed on day 9 (n-6/group). Body weight (B) and diarrhea (C) were recorded daily. (D) The entire intestinal length of each group of mice. (E) HE staining observed small bowel histological changes (scale bar: 100 μm). ELISA method was used to detect the expression of IL-6(F), TNF-alpha (G) and IL-1 beta (H) in each group. P <0.05, P <0.01, P < 0.001.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Tert-butyl hydroquinone, Tert-butyl hydroquinone, has a structure of

Example 1: TBHQ-reduced 5-FU-induced human intestinal epithelial cell injury

1. Experimental methods

In order to analyze the chemoprotective effect of TBHQ, human intestinal epithelial cells (HIEC cells) are firstly treated by adopting chemotherapeutics 5-fluorouracil (5-FU) with different concentrations, the optimal modeling concentration is selected to be 5 mu M5-FU, and half lethal dose of the HIEC cells occurs about 48 hours after the 5 mu M5-FU treatment (figure 1A). The HIEC cells were then pretreated with varying concentrations of TBHQ, with 5. mu.M of 5-FU added. After 48 hours, the amount of Lactate Dehydrogenase (LDH) released was measured using the CytoTox96 kit, and the cell viability was measured using the CCK-8 detection kit.

Apoptosis assay: HIEC cells were collected, stained with 7-AAD (2. mu.g/mL, Katriumyl, Jiangsu, China) for 20 min, and washed three times. A portion of the cells were then analyzed using a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA), and the data collected for analysis. Another portion of the cells was permeabilized using 0.5% Triton X-100(Sigma Aldrich, USA) for 2 minutes and washed three times. Subsequently, nuclei were stained with DAPI, then washed three times, and the number of 7-ADD positive cells was counted under a fluorescent microscope.

2. Results of the experiment

The results show that LDH release decreased in a TBHQ dose-dependent manner (fig. 1B), and cell viability increased in a TBHQ dose-dependent manner (fig. 1C). Meanwhile, it was found from the results of apoptosis and 7AAD cell staining by flow cytometry that the pretreatment concentrations of TBHQ were 10 μ M and 20 μ M, which showed good protection against cell damage and were close to each other, and 10 μ M was finally used at the cell level in view of cost saving (fig. 1D, 1E).

The experimental result shows that TBHQ has effective protection effect on 5-FU-induced human intestinal epithelial cell injury.

Example 2: TBHQ-reduced 5-FU-induced intestinal mucositis in mice

1. Experimental methods

The effect of TBHQ on 5-FU induced intestinal mucositis was studied in mice (figure 2).

The experimental procedure was as follows:

male C57/BL6 mice (8 weeks, 18-20g) were purchased from Duoduoshoo laboratory animals, Inc. (Sichuan, China). All mice were housed at 25 ℃ for a 12 hour light/dark cycle and kept in plastic cages with free access to food and water. Mice were randomized into three groups (n-6/group): a control group, a 5-FU group and a 5-FU + TBHQ group. The 5-FU group and the 5-FU + TBHQ group were each intraperitoneally injected with 50mg/kg5-FU for 5 days to induce intestinal mucositis. Meanwhile, 5-FU group and 5-FU + TBHQ group were intraperitoneally injected with DMSO or TBHQ (TBHQ was dissolved in DMSO, 10mg/kg) in an amount equal to each other once a day for 8 days (days 1 to 8). Body weight and diarrhea assessments were performed daily. On day 9, animals were sacrificed and intestinal length and HE staining histopathological analysis were recorded for all animals. Enzyme-linked immunosorbent assay (ELISA): mouse small intestine was collected and tested for IL-6, IL-1 β and TNF- α levels using ELISA kits (Mlbio, Shanghai, China) according to the instructions. All the above experimental protocols were approved by the institutional animal ethics committee.

2. Results of the experiment

The experimental results showed that TBHQ group significantly reduced diarrhea caused by 5-FU (fig. 2B), and significantly improved weight loss caused by 5-FU (fig. 2C). TBHQ administration significantly reduced the 5-FU-induced intestinal length loss compared to the 5-FU group (FIG. 2D). It was observed by histological, H & E staining analysis (fig. 2E) that TBHQ administration reduced pathological changes such as mucosal injury, necrosis, loss of tissue structure, edema and inflammatory cell infiltration (e.g. neutrophils) compared to the 5-FU group. In addition, TBHQ was found to significantly reduce the levels of IL-6, TNF-. alpha.and IL-1. beta. expression in mice induced by 5-FU when the serum inflammatory factor expression level in mice was measured by ELISA (FIG. 2F-H).

The experimental results show that TBHQ can obviously improve 5-FU induced intestinal mucositis, improve diarrhea, reduce intestinal length loss, relieve pathological changes (such as mucosal injury, necrosis, tissue structure loss, edema and inflammatory cell infiltration) and reduce the expression level of inflammatory factors.

In conclusion, the invention discovers for the first time that TBHQ has a protective effect on 5-FU-induced human intestinal epithelial cell injury, and tert-butyl hydroquinone can be used for preparing a medicine having a protective effect on human intestinal epithelial cell injury. The invention discovers for the first time that TBHQ can obviously improve 5-FU induced intestinal mucositis, can improve diarrhea, reduce intestinal length loss, relieve pathological changes (such as mucosal injury, necrosis, tissue structure loss, edema and inflammatory cell infiltration) and reduce the expression level of inflammatory factors. Therefore, TBHQ has good application prospect in preparing medicines for preventing and/or treating intestinal mucositis caused by chemotherapy.