CN111803477A - The use of disulfiram in the preparation of anti-head and neck cancer and anti-fibrosis drugs - Google Patents

Abstract

The invention discloses the use of disulfiram in the preparation of anti-head and neck cancer drugs and anti-pulmonary fibrosis drugs. The invention discovers for the first time that disulfiram inhibits the proliferation and promotes apoptosis of head and neck cancer cells, and proves that disulfiram is a potential clinical treatment drug for head and neck cancer. The head and neck cancer refers to head and neck cancer including oral and maxillofacial tumors (oral cancer, gum cancer, cheek cancer) and neck tumors. The present invention also finds for the first time that disulfiram has a relieving effect on pulmonary fibrosis, indicating that disulfiram has important significance in the clinical treatment of pulmonary fibrosis, and provides a new direction for the treatment of pulmonary fibrosis. The pulmonary fibrosis refers to idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis.

Description

The use of disulfiram in the preparation of anti-head and neck cancer and anti-fibrosis drugs

technical field

The invention relates to the technical field of drug development and application, in particular to the use of disulfiram (tetraethylthiuram disulfide) in the preparation of anti-head and neck cancer drugs and anti-pulmonary fibrosis drugs.

Background technique

Head and neck cancer is a tumor disease characterized by the surrounding of malignant epithelial cells in the oral cavity, pharynx, larynx and nasal cavity. Head and neck cancer is the 6th most common cancer. The 5-year survival rate of head and neck cancer patients is only 40%-50%. Every year, there are about 650,000 new cases of head and neck cancer in the world, and about 350,000 patients die from head and neck cancer every year. . Relevant studies have shown that smoking, alcohol consumption and papillomavirus infection are the main inducing factors of head and neck cancer. At present, the treatment procedures for head and neck cancer are very complicated, which will cause great economic and mental pressure to patients. At the same time, there is still a lack of effective head and neck cancer treatment methods and drugs.

Idiopathic pulmonary fibrosis is a pulmonary disease caused by progressive chronic lung inflammation and the outcome of the loss of pulmonary ventilation function. The incidence of idiopathic pulmonary fibrosis is mainly in adults, and the average survival period of patients is 3-6 years. Among the drugs discovered in recent years, pirfenidone and nintedanib are the only drugs that can be used for the treatment of pulmonary fibrosis, but so far the only effective method for treating pulmonary fibrosis is lung transplantation. Although research on idiopathic pulmonary fibrosis has been ongoing in recent years, effective drugs for pulmonary fibrosis are still lacking. The main cells involved in the pathogenesis of idiopathic pulmonary fibrosis include: lymphocytes, macrophages, dendritic cells, neutrophils, epithelial cells, endothelial cells and fibroblasts. The activated fibroblasts become myofibroblasts, and the activated fibroblasts secrete collagen, fibronectin and other substances, which lead to the deposition of extracellular matrix and eventually lead to pulmonary fibrosis.

Disulfiram was first used in the treatment of intestinal parasitic diseases. Later, it was found that the abnormality of alcohol tolerance in the treatment of patients was further studied and applied to abstain from symptoms such as alcoholism. The dosage of disulfiram is relatively high, the median lethal dose in animal experiments is 8.6g/kg, and the dosage of patients is generally 500mg per day. Therefore, the application of disulfiram to various diseases has high safety. As a small molecule drug, the application of disulfiram in head and neck cancer and pulmonary fibrosis has not been reported.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the use of disulfiram (tetraethylthiuram disulfide) in the preparation of anti-head and neck cancer drugs and anti-pulmonary fibrosis drugs; specifically in the preparation of anti-head and neck cancer (including oral and maxillofacial Use in medicaments and anti-pulmonary fibrosis medicines for oral cancer (oral cancer, gingival cancer, cheek cancer) and cervical cancer. The present invention discovers the therapeutic effect of disulfiram on pulmonary fibrosis for the first time. In the present invention, bleomycin, which produces pulmonary fibrosis side effects after clinical application, is selected as the inducer of experimental pulmonary fibrosis, so that the pulmonary fibrosis model is closer to the pathogenesis process of the human body, and the clinical applicability of the drug is stronger.

The purpose of this invention is to realize through the following technical solutions:

The present invention relates to the use of disulfiram in preparing medicine for treating and inhibiting head and neck cancer.

Preferably, the head and neck cancer is head and neck cancer including oral and maxillofacial tumors and neck tumors. The oral and maxillofacial tumors include oral cancer, gum cancer, and cheek cancer.

Preferably, the anti-head and neck cancer effect is achieved by promoting the apoptosis of head and neck cancer cells and inhibiting the proliferation of head and neck cancer cells.

Preferably, the disulfiram is used as an active ingredient, and pharmaceutically acceptable auxiliary materials and auxiliary ingredients are added to prepare a pharmaceutical preparation for use.

Preferably, the pharmaceutical preparation is selected from one of tablets, capsules, powders, granules, sprays, and sustained-release preparations.

The invention also relates to the use of disulfiram in the preparation of anti-pulmonary fibrosis drugs.

Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis.

Preferably, the disulfiram is used as an active ingredient, and pharmaceutically acceptable auxiliary materials and auxiliary ingredients are added to prepare a pharmaceutical preparation for use.

Preferably, the pharmaceutical preparation is selected from one of tablets, capsules, powders, granules, sprays, and sustained-release preparations.

Compared with the prior art, the medicine has the following beneficial effects:

1) The current anti-head and neck cancer drugs are very limited and have obvious drug side effects; disulfiram drugs are safe and have no serious adverse reactions; currently there are only two drugs for pulmonary fibrosis (nittanib and pirfenidone), and the drugs for pulmonary fibrosis are Disulfiram has been used for decades, with strong drug safety and no serious adverse reactions;

2) The production cost of the medicine is low, which is beneficial to clinical application.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 shows the results and statistics of the clone formation test; among them, A is the result of the clone formation experiment of HN4, HN12, HN30, Cal27, SCC9 and SCC25 cell lines after disulfiram stimulation, and B is the result of the clone formation experiment of HN4 cell line Statistical chart, C is the statistical chart of HN12 cell line clone formation experiment results, D is the statistical chart of HN30 cell line clone formation experiment results, E is the statistical chart of Cal27 cell line clone formation experiment results, F is the statistical chart of SCC9 cell line clone formation experiment results , G is the statistical graph of the experimental results of SCC25 cell line clone formation;

Figure 2 is the statistical chart of MTT test results; wherein, A is the statistical chart of HN4 cell line clone formation experiment results, B is the statistical chart of HN30 cell line clone formation experiment results, C is the statistical chart of Cal27 cell line clone formation experiment results, and D is HN12 Statistical chart of cell line clone formation experiment results;

Figure 3 is a flow cytometry result graph and a statistical graph of the cell cycle of head and neck cancer cells; wherein, A is a graph of the result of flow cytometry analysis of the cell cycle of HN30 cell line, and B is a graph of the statistic graph of the result of flow cytometry analysis of the cell cycle of HN30 cell line;

Figure 4 is the flow cytometry result graph and statistical graph of cell apoptosis; wherein, A is the flow cytometry analysis result of HN30 cell line cell apoptosis, and B is the statistic graph of the cell apoptosis flow cytometry analysis result of HN30 cell line;

Figure 5 is a schematic diagram of H&E staining of fixed mouse lung tissue;

Figure 6 is a schematic diagram of Masson staining of fixed mouse lung tissue;

Figure 7 is a schematic diagram showing the level of fibronectin mRNA detected by quantitative RT-PCR method after total RNA was extracted from mouse lung tissue.

Figure 8 is a schematic diagram showing the level of collagen mRNA detected by quantitative RT-PCR method after extracting total RNA from mouse lung tissue;

FIG. 9 is a schematic diagram showing the results of measuring hydroxyproline content in lung tissue of mouse lung tissue by using a hydroxyproline detection kit.

Detailed ways

The present invention will be described in detail below with reference to the embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

The disulfiram structural formula used in the present invention is as follows:

Example 1. Study on the function regulation effect of disulfiram on head and neck cancer cells

Disulfiram can significantly inhibit the proliferation of human head and neck cancer cells and promote the apoptosis of human head and neck cancer cells. It provides an important basis for clinical treatment of head and neck cancer. This example further demonstrates the specific action mechanism of disulfiram on head and neck cancer cell lines, and also provides the possibility for disulfiram on related diseases with similar molecular mechanisms.

1 Experimental material

1.1 Cell lines: HN30 cell line, Cal27 cell line, HN4 cell line, HN12 cell line, SCC9 cell line and SCC25 cell line.

1.2 Drugs and reagents: MTT detection kit (Biyuntian), apoptosis detection kit (Biyuntian).

1.3 Main equipment: BD LSRFortessa flow cytometer; multi-function microplate reader;

2 Methods and results

2.1 Experimental method

2.1.1 Clone formation experiment:

Controlling the cells to logarithmic growth phase, the head and neck cancer cell line used in the experimental study was HN4. The HN4 cell line is an adherent cell. Take the cell culture dish out of the incubator, operate aseptically in the ultra-clean bench, discard the supernatant, and wash twice with PBS to remove the residual medium. Add 1ml of trypsin to the culture dish. When the cells are observed in a circular shape under the microscope, then add 3ml of DMEM medium containing 10% FBS to them for neutralization. After the digestion, add the digested cells to the corresponding centrifuge tube. After centrifuging at a low speed of 1000rpm for 3min, aspirate the supernatant, add 10ml of DMEM medium containing 10% FBS to the centrifuge tube, and gently pipet the cells with a 1ml pipette tip, so that the cells are evenly dispersed in the medium . Take 10 μl of the evenly dispersed cell suspension for counting, and calculate the cell density according to the formula: cell density=number/4×10 ⁴ cells/ml. The volume of inoculated cell suspension in each well is 0.1 ml, and after inoculation, ensure that the cells are kept in a uniform state. Each well is guaranteed to have 200 cells. Add 100 μl of DMEM medium containing 10% FBS to it, and shake it properly to ensure uniformity. Cells spread out. Add disulfiram at concentrations of 0, 0.05, 0.10, 0.20 μM. The 96-well plate was tapped gently to ensure dispersion, followed by incubation at 37°C for 7 days. During this process, observe closely, take out the well plate when cell clusters are found, discard the supernatant, add 200 μl methanol to each well of the well plate for fixation, let stand at room temperature for 30 minutes, and rinse gently with pure water After treatment, 100 μl of Gimsa staining solution (1) was added to each of them, and the mixture was properly mixed and placed at room temperature for 20 min. Continue to add 200 μl of Gimsa staining solution (2), and leave it at room temperature for 30 min. After sufficient reaction, then ensure that the cells are stained, aspirate the staining solution, and perform a sufficient washing operation, and then determine the number of clones in each well. The obtained results were recorded, and the compounds that inhibited tumor proliferation were recorded for re-screening.

2.1.2 MTT experiment

(1) Preparation: During this operation, first prepare MTT stock solution according to the requirements, accurately weigh 50 mg of MTT powder, and keep away from light. During the operation, all the powder was added to a 10 ml sterile centrifuge tube, and after ultrasonication, the powder was completely dissolved in sterile deionized water. In the sterilization process, the method of filtration and sterilization is generally selected, and the 0.22 μM membrane is used for filtration. The corresponding filtrate is transferred to a sterile centrifuge tube, and then 1ml is removed and dispensed into a centrifuge tube. Store properly at 80°C.

(2) Experimental procedure: HN12, HN4, Cal27 and HN30 cells were plated in wells, digested and then resuspended in DMEM medium containing 10% FBS, and resuspended after appropriate pipetting. The density of the hemocytometer was controlled to be 1×10 ⁵ cells/ml. After appropriate pipetting, the 96-well plate was taken out, and 0.1 ml of cell suspension was added to the target well. During the experiment, 1×10 ⁴ cells were added to each group (the number of wells to be plated is determined according to the proliferation ability of different cells), no cells were added to the surrounding wells, and sterilized phosphate buffer was added to effectively prevent the liquid from evaporating. . Appropriately spray alcohol on the surface of the well plate and then transfer it to the incubator for adherence and growth. After culturing for about 12 hours, it was observed that the cells were able to cover the bottom of the well plate and were taken out when 80% was reached. After discarding the supernatant, 0.2 ml of fresh medium was added to each well, and 0, 16, 32 and 64 μM of disulfiram were added to each well. The concentration has six duplicate wells. Gently tap the side of the 96-well plate to disperse the drug in the medium as evenly as possible. After sufficient incubation for 24 hours, the well plate is taken out, and 0.01 ml of MTT solution is added to each well under aseptic conditions. Then put the plate back into the cell culture incubator and continue to incubate for 4 hours. Use a 1ml syringe (the bevel of the syringe needle is clamped with forceps to make the needle flat) to gently aspirate the supernatant, and then pass through the corresponding dimethyl sulfoxide. After adding 0.1ml to each well, it was shaken for 10min on a shaking table, and tapped gently to ensure that the crystals could be completely dissolved in DMSO, and then the absorbance was detected. The absorbance of the data was processed and analyzed.

(3) Calculate the tumor cell growth rate or tumor cell growth inhibition rate according to the following formula:

Cell growth rate=(OD administration group/OD solvent group)×100%

Cell growth inhibition rate=(1-OD administration group/OD solvent group)×100%

2.1.3 Cell cycle detection

Experimental steps for detecting cell cycle distribution with PI dyes:

Resuscitate the cells and adjust the cell state so that it is in the logarithmic growth phase. At this time, the cell state is the best. After collecting the corresponding cells, adjust the cell density and inoculate 1×10 ⁶ cells in each well of the six-well plate. Each well was inoculated with 1 ml of cell suspension, and then supplemented with 1 ml of DMEM medium containing 10% FBS. After culturing overnight, the supernatant was discarded and the compound of the corresponding concentration was added to the drug for 24 hours. Digest with 0.25% trypsin, centrifuge at 1000rpm in a low-speed centrifuge to separate the supernatant, and then add 1ml of pre-cooled PBS to each tube to wash the cells twice to remove the residual trypsin, etc., after the same centrifugation Remove the supernatant liquid, continue to add 0.5ml of PBS to each tube, and gently suspend the cell pellet with a 1ml pipette tip to disperse the cells in a single-cell state. 35ml of absolute ethanol and 15ml of deionized water were mixed evenly, and placed in a pre-cooled at -20°C) and added to the flow tube. To ensure the dispersion of cells, 4 ml of pre-cooled 70% ethanol should be added to each flow tube. , uniformly disperse the cell suspension into 70% ethanol with a pipette tip for fixation. This step is more important. Rotate the flow tube while adding the cell suspension, and continue to pipet the liquid to completely disperse the cells into single cells in 75% ethanol. , and then placed at 4°C, or -20°C for at least 24 hours, centrifuged at 1500 rpm for 5 min before flow cytometry, and then discarded the supernatant to remove ethanol. To further remove residual ethanol, cells need to be washed with PBS Precipitate, also after centrifugation, discard the supernatant, and gently resuspend the remaining cell pellet with 400 μl PBS. In this step, it is necessary to ensure that the cells are completely single-celled to reduce the experimental error, and then add RNase ( RNase) and then placed in room temperature for 30min to completely remove the RNA in cells (the concentration of RNase used is 10μg/ml), after 30min, directly add the prepared PI dye (final concentration 50μg/ml) to each tube, room temperature The cell cycle distribution and changes were detected on a flow cytometer after being placed in the dark for 15 minutes. Since the PI dye itself is relatively sticky, it is necessary to remove the adhered cells during detection. The data were analyzed with Flowjo 7.6.

2.1.4 Detection of apoptosis

Annexin V-FITC/PI double staining steps:

The cultured HN30 cells were in logarithmic growth phase, digested with 0.25% trypsin, centrifuged at 1000 rpm for 3 min, removed the supernatant, resuspended with an appropriate amount of DMEM medium containing 10% FBS, counted on a hemocytometer, and plated wells, generally six wells Plate 6×10 ⁵ , 12-well plate 3×10 ⁵ , overnight culture. When the cells proliferated to 70%-80%, the orifice plate was replaced with a new culture medium, and the corresponding drugs were added for treatment for 24h. After labeling the flow tubes, collect the supernatant medium, wash once with PBS (to remove residual serum in the medium), and collect PBS into the corresponding centrifuge tubes containing apoptotic cells, lower layer of adherent cells After the collection by trypsinization, the supernatant was combined with the supernatant, and then centrifuged at low speed for 3 min. Then, the supernatant was appropriately washed with phosphate buffer, and the supernatant was discarded. After the low-speed centrifugation was continued, 500 μl of 1×Binding Buffer was added to each tube to resuspend the cells ( Dilute 5× Binding Buffer with double-distilled water to 1× working solution), centrifuge the Annexin-FITC staining solution and PI staining solution in the apoptosis detection kit briefly, and store them on ice temporarily for later use. After the pipetting, part of the cell suspension was separated from each tube for control study. PI 6μl) group, the purpose of setting three groups of control groups is to adjust the compensation generated between the fluorescence during the on-board detection, adding 5μl Annexin V-FITC and 10μl PI to each tube. After appropriate mixing, it was then incubated at room temperature for 5 min. The FITC detection channel was used to detect the two, and the results were analyzed to determine the apoptosis of the cells.

2.2 Experimental results

2.2.1 The effect of drugs on the proliferation of head and neck cancer cells

HN4, HN12, Cal27, HN3, SCC9 and SCC25 head and neck cancer cells were plated in 6-well plates,

Figure 1: Antitumor activity of disulfiram. Different concentrations of disulfiram (0, 0.05, 0.10, 0.20 μM) were used to perform clonogenic experiments on various head and neck cancer cell lines (HN4, HN12, Cal27, HN3, SCC9 and SCC25 cells) (Fig. 1A), while the effect of disulfiram on the colony formation of each head and neck cancer cell was counted and the corresponding statistical graphs were drawn (Fig. 1B-G). The experimental and statistical results showed that the effect of disulfiram on the above cells The proliferation function has a certain inhibitory effect, and the inhibitory effect is dose-dependent. Among them, the inhibitory effect of HN30 was the most obvious.

2.2.2 The effect of disulfiram on cell viability of head and neck cancer cell lines

Figure 2: Different concentrations (0, 16, 32, and 64 μM) of disulfiram were used to treat cells for 24 hours. The MTT assay was used to detect the effect of drugs on cell activity, and the cell proliferation rate was calculated. Figure 2A shows HN4 cell line, and Figure 2B shows HN30 Figure 2C is the Cal 27 cell line, and Figure 2D is the HN12 cell line. The experimental statistics show that disulfiram has a certain toxic effect on the above cells, and the killing effect is concentration-dependent.

2.2.3 The effect of disulfiram on the cycle of head and neck cancer cell lines

Figure 3: The process of cell division is divided into the cell division phase (M phase) and the interphase. The cells in the interphase are further divided into the early DNA synthesis phase (G0/G1 phase), the DNA synthesis phase (S phase) and the late DNA synthesis phase (G2). Propidium iodide (PI) staining can bind DNA in cells. Since the DNA content of intracellular DNA is different in different stages of division, we detect the division stages of cells by PI staining, using different concentrations (0, 10, 30 and 100 μM) of disulfiram was treated in cells for 24 h, and the distribution of HN30 cell cycle was detected by flow cytometry, and statistical analysis was performed. The experimental statistical results show that disulfiram can make HN30 head and neck cancer cells stagnate in the S phase and G2/M phase of cell proliferation, thereby effectively blocking the proliferation process of head and neck cancer cells. The blocking effect is dose-dependent.

2.2.4 Disulfiram-induced apoptosis

Figure 4: A: Annexin V-FITC/PI detects the apoptosis of HN30 cells treated with different concentrations of disulfiram (0, 10, 30 and 100 μM) for 24 hours, and the proportion of apoptotic cells is counted; the statistical graph of the experimental results shows , disulfiram can promote the apoptosis process of head and neck cancer cells, and it can significantly promote cell apoptosis when the concentration is 30 μM.

The research results of this example show that disulfiram has a significant inhibitory effect on the proliferation of head and neck cancer cells, and has a promoting effect on the apoptosis of head and neck cancer cells, indicating that disulfiram has a potential role and value in clinical application of head and neck cancer. It provides a new direction for the search for anti-head and neck cancer drugs.

Example 2. Study on the regulating effect of disulfiram on pulmonary fibrosis

In this experiment: the preparation method of disulfiram used in animal experiments is as follows, disulfiram is dissolved in DMSO, the concentration is 2g/ml, then 25μl is mixed with 400μl ethanol and 400μl castor oil, and then mixed with 7.2ml PBS, The resulting mixture was sonicated to obtain a suspension.

The preparation method of disulfiram used in cell experiments: disulfiram was directly dissolved in DMSO to prepare two stock solutions of 100 mM and 10 mM, and stored in a -80°C refrigerator.

In the present embodiment, it is found through animal experiments that disulfiram has a certain therapeutic effect on pulmonary fibrosis, and the test method and the results are as follows:

1 Experimental material:

1.1 Experimental animals: C57BL/6 mice, male, weighing about 25 g, were raised in the Experimental Animal Center of Shanghai Jiaotong University, air-conditioned at room temperature of 20-25 °C, sterile water, and sterilized rat food.

1.2 Reagents: disulfiram (Disulfiram, Sigma), castor oil (Sangon Bio), anhydrous ethanol (Sangon Bio).

1.3 Main equipment: Real-time PCR system (ABI), upright microscope (Olympus).

2 Methods and results

2.1 Experimental method

2.1.1 Construction of mouse pulmonary fibrosis model

23 mice were randomly divided into 4 groups and were given bleomycin or PBS and disulfiram or solvent control respectively (6 mice were given bleomycin for disulfiram and solvent control group, and PBS was given to solvent control group) 5, and 6 in the disulfiram group given PBS). On the 0th day of modeling, mice were given bleomycin (tracheal administration), each mouse was given bleomycin by body weight, and bleomycin was dissolved in PBS to prepare a working concentration (1.4U/kg), And tracheal administration was performed, and the day of modeling was set as day 0. The mice were intraperitoneally injected with 50 mg/kg disulfiram every day, and the lung tissue and peripheral blood of the mice were collected for analysis after 21 days.

2.1.2 HE staining of lung tissue: After the lung tissue was fixed in paraformaldehyde, it was routinely dehydrated, embedded, and sectioned. After deparaffinization, the Biyuntian H&E staining kit was stained and stained according to the instructions, and finally mounted for observation.

2.1.3 Masson staining of lung tissue: The lung tissue was routinely dehydrated, embedded, and sectioned after paraformaldehyde fixation. After deparaffinization, Nanjing built Masson staining kit for staining and staining according to the instructions, and finally mounted for observation.

2.1.4 Detection of hydroxyproline in lung tissue: After the lung tissue was taken out from the ultra-low temperature refrigerator, the changes in hydroxyproline level were detected after being processed according to the instructions of the hydroxyproline detection kit of Nanjing Jiancheng Biological Company.

2.1.5 Lung tissue mRNA extraction, reverse transcription and qPCR experiments: Lung tissue was cryogenically ground, treated with chloroform, isopropanol and 75% ethanol, and centrifuged to obtain total RNA, which was performed using the toyobo reverse transcription kit according to the instructions After reverse transcription, the obtained cDNA was detected by real-time quantitative PCR with toyobo SYBR Green kit.

2.2 Experimental results

2.2.1 Pathological analysis of lung tissue

Due to bleomycin-induced pulmonary fibrosis, the lung tissue of mice will have lesions caused by excessive repair, and the lesions will have the deposition of extracellular matrix such as collagen. We analyzed histopathological sections (H&E staining and Masson staining) of mouse lungs.

The mouse lung tissue was fixed, dehydrated, embedded, sectioned and stained. The results of H&E staining showed (Figure 5) that after bleomycin modeling, the lung tissue had a lesion area, while the lung tissue of the disulfiram-administered group had lesions. Areas were significantly reduced, indicating the mitigating effect of disulfiram on pulmonary fibrosis. Masson staining (Fig. 6) reflected the deposition of collagen in lung tissue. The results showed that collagen deposition occurred in lung tissue after bleomycin modeling, and disulfiram could attenuate the deposition of collagen caused by pulmonary fibrosis.

2.2.2 RT-PCR detection of mRNA and protein levels of pulmonary fibrosis marker molecules in lung tissue

During the process of pulmonary fibrosis, the expression of Fibronectin and Collagen in mouse lung tissue will increase. We detected the mRNA levels of Fn1 and Col1a1 in lung tissue. The results are shown in Figures 7 and 8. After modeling, the total RNA of mouse lung tissue was extracted and then reverse transcribed, followed by real-time PCR to detect the changes of Fn1, Col1a1 mRNA levels; at the same time, because hydroxyproline is specific for collagen components in lung tissue As an amino acid, we detected the content of hydroxyproline in mouse lung tissue (Figure 9). The above experimental results show that disulfiram can significantly inhibit the expression of pulmonary fibrosis-related molecules.

Example 3

Preparation of tablets:

Take 500 g of disulfiram, add an appropriate amount of starch to make granules, add an appropriate amount of magnesium stearate, mix well and compress into 1000 tablets, one tablet once a day.

Example 4

Preparation of capsules

Take 500g of disulfiram, add an appropriate amount of diluent, mix well, and fill it into empty capsules. Make 1000 capsules, 1 time a day, 1 capsule each time.

Example 5

Preparation of granules

Take 40g of disulfiram, add 670g of sucrose and 290g of auxiliary dextrin, mix well, and use a roller flat granulator to granulate to obtain 1000g of disulfiram granules, 1 pack (10g) each time, 3 times a day.

The results of this experimental study show that the disulfiram of the present invention has a significant inhibitory effect on bleomycin-induced pulmonary fibrosis, indicating that disulfiram has an important clinical value for pulmonary fibrosis, and is an anti-fibrosis therapeutic drug The research and development provides a new direction.

The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

Claims (9)

1. Use of disulfiram in the preparation of a medicine for treating and inhibiting head and neck cancer. 2 . The use according to claim 1 , wherein the head and neck cancer is head and neck cancer including oral and maxillofacial tumors and neck tumors. 3 . 3 . The use according to claim 2 , wherein the oral and maxillofacial tumors include oral cavity cancer, gum cancer, and buccal cancer. 4 . 4. The use according to any one of claims 1 to 3, wherein the disulfiram is used as an active ingredient, and pharmaceutically acceptable adjuvants and auxiliary ingredients are added to prepare a pharmaceutical preparation for use. 5. The use according to claim 4, wherein the pharmaceutical preparation is selected from the group consisting of tablets, capsules, powders, granules, sprays, and sustained-release preparations. 6. A use of disulfiram in the preparation of an anti-pulmonary fibrosis drug. The use according to claim 6, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis and clinical secondary pulmonary fibrosis. 8. The use according to claim 6 or 7, characterized in that, using the disulfiram as an active ingredient, adding pharmaceutically acceptable adjuvants and auxiliary ingredients to prepare a pharmaceutical preparation for use. 9. The use according to claim 8, wherein the pharmaceutical preparation is selected from one of tablets, capsules, powders, granules, sprays, and sustained-release preparations.

Images