Anticancer suppository for co-releasing chemotherapeutic drug and hydrogen molecule and application thereof

Abstract

The invention relates to an anti-cancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules and application thereof, provides a new strategy for relieving side effects and improving curative effects by locally cooperating with hydrogen chemotherapy based on anti-cancer selectivity of hydrogen and a protection effect on normal cells, and uses fatty glyceride (FAG) to wrap 5-fluorouracil (5-FU, FG), so that the anti-cancer suppository is prepared. A novel anti-cancer rectal suppository (5-FU / CSN-FAG) is constructed from 5-FU, a first-line drug for treating colorectal cancer and cerium silicide nanoparticles (CSN), and the novel anti-cancer rectal suppository (5-FU / CSN-FAG) has the behavior of continuous hydrolysis to release hydrogen and is synchronous with release of 5-FU. The 5-FU / CSN-coated FAG suppository is used for treating colonic tumors once a day for continuous three weeks, tumor recurrence is avoided after drug withdrawal, and intestinal tracts can be effectively protected from chemotherapy injury. In terms of mechanism, H2 generated by the CSN reduces toxicity of 5-FU to normal cells of intestinal tracts by removing over-expressed reactive oxygen species (ROS) and correcting energy metabolism, inhibits respiration of colon tumor cells through a CO signal channel, and assists the 5-FU in promoting apoptosis of the colon tumor cells. And due to high biological safety and treatment effectiveness, the developed suppository has very high clinical transformation potential.

Description

Technical Field

[0001] The invention relates to a medicine for treating colorectal cancer and application of the medicine in treating colorectal cancer. Background Art

[0002] Colorectal cancer is recognized as the third most common malignant tumor in the world. In 2022, there were more than 1.9 million new cases of colorectal cancer and 904,000 deaths, ranking third in incidence and second in mortality. It is estimated that the number of new cases will increase to 2 million to 5 million in 2035. At present, the main treatment strategies for colorectal cancer include surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Early cancer treatment is mainly based on surgical resection, while chemotherapy and radiotherapy as adjuvant therapy can reduce the risk of tumor recurrence and inhibit tumor growth, but often bring unbearable toxic side effects. The conventional administration of chemotherapy drugs is generally intravenous injection and oral administration, but it has serious side effects on the whole body, especially the intestine. Chemotherapy suppositories are considered to be a promising dosage form, which can increase the local drug concentration at the tumor site, prolong the time of drug accumulation in the tumor, enhance the efficacy of the drug, and avoid the toxic side effects of chemotherapy drugs on distal organs. Studies have shown that 5-FU suppositories do not cause systemic toxicity to mice, while 63% of mice in the 5-FU intravenous injection group developed symptoms such as diarrhea, weight loss and bone marrow suppression 10 days after administration. However, the case of preoperative radiotherapy combined with 5-FU suppository treatment showed that when the local concentration of chemotherapy drugs in the rectum is too high, it will also cause toxic side effects such as tenesmus and anal pain in the intestinal tissue. Therefore, reducing the intestinal toxicity of high-concentration chemotherapy drugs in the colorectum is of great significance to improving the effect of chemotherapy, but it is still a difficult problem.

[0003] Hydrogen molecule (H2) is a therapeutic gas with high biosafety. Its broad-spectrum anti-inflammatory and selective anti-tumor effects have been confirmed by extensive basic research and clinical trials. Taking advantage of these characteristics, oral hydrogen-rich water / inhaled hydrogen combined with chemotherapeutic drugs can enhance the anticancer effect of chemotherapeutic drugs and reduce the toxic side effects of chemotherapeutic drugs on normal cells / tissues. The combination of hydrogen-rich water and the chemotherapeutic drug 5-FU can inhibit the growth of colorectal cancer and improve the survival rate of mice. Daily intake of 1L of hydrogen-rich water can significantly reduce the liver damage caused by mFOLFOX6 chemotherapy in colorectal cancer patients. However, these traditional hydrogen administration routes have low bioavailability at the colorectal tumor site, resulting in limited effectiveness of hydrogen-chemotherapy combination therapy (hydrogen chemotherapy). The co-delivery of H2 and chemotherapeutic drugs can be achieved by injecting H2-releasing nanomaterials such as iron porphyrin metal organic framework (Fe-MOF) nanocrystals, which enhances the accumulation of H2 in tumors, but the tumor-targeted delivery efficiency of nanomaterials is quite low. Therefore, site-specific delivery and sustained release of H2 at the colorectal tumor site are essential for effective adjuvant chemotherapy. Summary of the invention

[0004] Based on the above analysis, the present invention proposes a technical solution for local synergistic hydrogen chemotherapy of colorectal tumors using suppositories co-loaded with hydrogen-releasing nanomaterials / chemotherapeutic drugs. The technical solution is as follows:

[0005] The first aspect of the present invention provides an anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules. The drug suppository is composed of chemotherapeutic drugs, hydrogen-producing materials and thermosensitive matrix materials. The mass ratio of the chemotherapeutic drugs, hydrogen-producing materials and thermosensitive matrix materials is 1-20:5-50:50-500.

[0006] In some embodiments, the chemotherapy drugs include but are not limited to 5-fluorouracil, capecitabine, S-1, raltitrexed, oxaliplatin, and irinotecan.

[0007] In some embodiments, the thermosensitive matrix material includes but is not limited to cocoa butter, balsam pear butter, Chinese tallow fat, semi-synthetic coconut oil esters, semi-synthetic lycopene oil esters, semi-synthetic palm oil esters, mixed fatty acid glycerides, semi-synthetic fatty acid esters, hydrogenated vegetable oils, glycerol gelatin, polyethylene glycols, polyoxyethylene (40) monostearate, and poloxamer, and has thermosensitive properties with a solid-liquid transition temperature of 34-38 degrees Celsius.

[0008] In some embodiments, the hydrogen-producing material includes but is not limited to CeSi2, MgH2, CaH2, MgSi2, preferably nanoparticles whose hydrogen release rate is synchronized with the chemotherapeutic drugs, such as nanoparticles with a particle size of 50-300 nm, which can release most of the hydrogen within 24 hours.

[0009] In some preferred embodiments, the novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules comprises, by weight, 10 parts of cerium silicide, 2.5 parts of 5-FU, 1 part of carbomer, and 100 parts of fatty acid glycerides.

[0010] The second aspect of the present invention provides an application of the novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules in the field of intestinal tumor treatment. The intestinal tumor is a colorectal tumor. The novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules provided by the present invention completely melts within 30 minutes at body temperature and can stay in the colorectum for 24 hours.

[0011] The novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules provided by the present invention can realize the synchronous sustained release of H2 and 5-FU in the colorectum.

[0012] The novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules provided by the present invention can completely eradicate colorectal tumors within 3 weeks.

[0013] The novel anticancer suppository for co-releasing chemotherapeutic drugs and hydrogen molecules provided by the present invention can avoid the recurrence of colorectal tumors after drug withdrawal and reduce the toxic and side effects of chemotherapeutic drugs.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the local sustained release of 5-FU and H2 in the colon mediated by 5-FU / CSN@FAG suppositories, and the mechanism by which H2 enhances the anticancer efficacy of 5-FU and reduces the side effects of 5-FU on normal colon cells;

[0016] Figure 2 Characterization of CSN and 5-FU / CSN@FAG suppository, where a is the SEM image of CSN, b is the size distribution of CSN measured by DLS, c is the digital image of 5-FU / CSN@FAG suppository heated at 25°C and 37°C for different time periods, d is the melting curve of 5-FU / CSN@FAG suppository, e is the digital image and fluorescence image of intestinal tissue extracted in vitro after administration of Cy5-labeled 5-FU / CSN@FAG suppository at different time periods, and f is the corresponding quantitative analysis of colon fluorescence intensity;

[0017] Figure 3 The XRD data of raw materials CeSi2 powder and cerium silicide nanoparticles (CSN);

[0018] Figure 4 SEM images of CeSi2 raw powder, CSNs with diameters of about 300 nm and 50 nm (a), and the change of hydrogen release concentration over time in simulated intestinal fluid (b);

[0019] Figure 5 FT-IR spectra of cerium silicide nanoparticles (CSN), carbomer, 5-FU, FAG, and 5-FU / CSN@FAG suppositories;

[0020] Figure 6 Raman spectra of cerium silicide nanoparticles (CSN), carbomer, 5-FU, FAG, and 5-FU / CSN@FAG suppositories;

[0021] Figure 7 is the melting point of FAG suppository and 5-FU / CSN@FAG suppository detected by DSC;

[0022] Figure 8 Digital images of blank control, 5-FU, CSN, and 5FU+CSN, as well as 5-FU / CSN@FAG suppositories gradually melted at 37°C;

[0023] Fig. 9 This is a melting point determination curve of 5-FU / CSN@FAG suppositories after one year of storage;

[0024] Fig.10 This is the XRD data diagram of CSN extracted from 5-FU / CSN@FAG suppository after storage for one year;

[0025] Fig.11 The UV absorption spectra of 5-FU solutions with different fixed concentrations and the corresponding standard curve at 265 nm;

[0026] Fig.12 This is a graph showing the change of 5-FU concentration in the blood over time after taking 5-FU / CSN@FAG suppositories;

[0027] Fig.13 The toxicity diagram of CSN and 5-FU in 5-FU / CSN@FAG suppository at different ratios on HCT116 tumor cells;

[0028] Fig.14 The 5-FU and H2 release behaviors of 5-FU / CSN@FAG suppositories are shown: a and b respectively show the H2 and 5-FU release behaviors of 5-FU / CSN@FAG suppositories under 100 rpm shaking conditions in simulated colonic fluid at 37°C, c and d show the distribution and metabolism of 5-FU-FITC / CSN@FAG suppositories in the intestine after in vitro fluorescence imaging and the corresponding average fluorescence intensity of the colon, e is the concentration of 5-FU in the colon and small intestine of mice after administration of 5-FU / CSN@FAG suppositories, and f is the H2 concentration in the colon of mice measured by hydrogen electrode at 0, 1, 2 and 6 h after administration of 5-FU / CSN@FAG suppositories;

[0029] Fig.15 The results show the mechanism of enhancing the efficacy and reducing the toxicity of hydrogen chemotherapy combined with chemotherapy: a is a schematic diagram of the in vitro cytotoxicity measurement method of 5-FU / CSN@FAG suppositories; b and c are the cytotoxicity of 5-FU and H2 released from 5-FU / CSN@FAG suppositories with different CSN to 5-FU mass ratios on NCM460 cells and CT26 cells, respectively; d is the mechanism of H2 enhancing the toxicity of 5-FU on normal NCM460 cells; e and f are the confocal images of mtROS (red fluorescence) and Hoechst (blue fluorescence) staining of NCM460 cells after different treatments and the corresponding quantitative analysis of ROS levels; g is the ATP level in NCM460 cells after different treatments; h is the mechanism of H2 enhancing the toxicity of 5-FU on colon cancer CT26 cells; ik is the confocal image of COP-1 (green fluorescence) staining of CT26 cells after different treatments with Hoechst (blue fluorescence) (i) and the corresponding quantitative analysis of CO content (j) and ATP level (k) in CT26 cells after different treatments;

[0030] Fig.16Fluorescence images of live / dead NCM460 cells stained with Calcein-AM / PI incubated with 5-FU / CSN@FAG suppositories with different CSN to 5-FU ratios;

[0031] Fig.17 Fluorescence images of live / dead CT26 cells stained with Calcein-AM / PI incubated with 5-FU / CSN@FAG suppositories with different CSN to 5-FU ratios;

[0032] Fig.18 Fluorescence images of HCT116 live / dead cells stained with Calcein-AM / PI incubated with 5-FU / CSN@FAG suppositories with different CSN to 5-FU ratios;

[0033] Fig.19 The cytotoxic effect of CSN@FAG with different CSN concentrations on NCM460 cells;

[0034] Fig. 20 Fluorescence images of live / dead NCM cells stained with Calcein-AM / PI incubated with different concentrations of CSN@FAG suppositories;

[0035] Fig.21 The cytotoxicity of CSN@FAG with different CSN concentrations on CT26 and HCT116 cells;

[0036] Fig. 22 Fluorescence images of live / dead CT26 cells incubated with different concentrations of CSN@FAG suppositories stained with Calcein-AM / PI;

[0037] Fig.23 Fluorescence images of HCT116 live / dead cells incubated with different concentrations of CSN@FAG suppositories stained with Calcein-AM / PI;

[0038] Fig.24 The DNA damage images of CT26 treated with CSN suppositories, 5-FU suppositories and 5-FU+CSN suppositories (a) and the corresponding fluorescence intensity statistics of DNA damage (b);

[0039] Fig.25 The graph shows the changes in ATP levels in HCT116 cells treated with CSN suppositories, 5-FU suppositories, and 5-FU+CSN suppositories at different time points;

[0040] Fig.26The results of hydrogen chemotherapy for orthotopic colon cancer in mice are as follows: schematic diagram of colon cancer model establishment and treatment (a), bioluminescent imaging monitoring of colon tumor at fixed time points (the red arrow indicates that drug administration was stopped at the time when the tumor disappeared) (b), survival curves of mice in different treatment groups (c), changes in tumor volume in different treatment groups: control group (d), matrix FAG suppository group (e), CSN@FAG suppository group (f), 5-FU@FAG suppository group (g), 5-FU / CSN@FAG suppository group (h);

[0041] Fig. 27 This is a graph showing the changes in mouse body weight during treatment in different groups;

[0042] Fig.28 The figure shows the pathological analysis of colon tissues of mice with tumors treated with hydrogen chemoembolization, including: H&E staining images of colon tissues and magnified images of corresponding typical tumor areas (red dashed line boxes) and normal colon areas (blue dashed line boxes) (ac), immunofluorescence staining of Ki67 (d) and TUNEL (e) in tumor areas and corresponding fluorescence intensity statistics (f), immunofluorescence staining of IL-6 (g) and TNF-α (h) in colon tissues and corresponding fluorescence intensity statistics (i);

[0043] Fig.29 This is a picture of the anus of mice after one week of once-daily suppository administration;

[0044] Fig.30 The results of rectal H&E staining of mice in each group after one week of once-daily suppository administration;

[0045] Fig.31 H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of mice after treatment;

[0046] Fig.32 Figure 2 shows liver / kidney function, blood and heart function parameters of mice treated with different doses (n=3) of CSN suppositories.

[0047] Fig.33 Figure 2 shows liver / kidney function, blood and heart function parameters of mice treated with 5-FU suppositories at different doses (n=3);

[0048] Fig.34 Figure 2 shows liver / kidney function, blood and heart function parameters of mice treated with 5-FU+CSN suppositories at different doses (n=3);

[0049] Fig.35 The release behaviors of Cape and H2 from Cape / MSN@PEG suppositories: a and b respectively show the release behaviors of H2 and Cape from Cape / MSN@PEG suppositories under 100 rpm shaking in simulated colonic fluid at 37°C;

[0050] Fig.36 The results show the synergistic and toxicity-reducing mechanism of hydrogen chemotherapy combined with chemotherapy: a, b and c are the cytotoxicity of Cape and H2 released from Cape / MSN@PEG suppositories on NCM460 cells (normal cells), CT26 cells and HCT116 cells (cancer cells), respectively. DETAILED DESCRIPTION

[0051] The present invention is further described in detail and completely below in conjunction with the embodiments, but the contents of the present invention are not limited thereto.

[0052] The present invention proposes a Figure 1 The figure shows a strategy for local synergistic hydrogen chemotherapy of colorectal tumors using suppositories co-loaded with hydrogen-releasing nanomaterials / chemotherapeutic drugs. The figure shows a schematic diagram of the local sustained release of 5-FU and H2 in the colon mediated by 5-FU / CSN@FAG suppositories, as well as the mechanism by which H2 enhances the anti-cancer efficacy of 5-FU and reduces the side effects of 5-FU on normal colon cells.

[0053] Example 1

[0054] 1. Preparation and characterization of CSN and 5-FU / CSN@FAG suppositories

[0055] The CeSi2 raw material powder is crushed into nanoparticles with an average particle size of about 200nm to 900nm, or 200-500nm, preferably 300nm, by ultrasonic crushing. Figure 3 As shown in the SEM image in a and the DLS data in b, Figure 2 From the XRD data in Figure 2, CSN maintains the crystal structure of CeSi2. Compared with the raw material CeSi2 powder, cerium silicide nanoparticles (CSN) show higher hydrogen production efficiency. Figure 4 As shown in the hydrogen electrode measurement in , CeSi2 raw powder, CSN with a particle size of about 300nm and 50nm reached the peak of hydrogen release rate in simulated intestinal fluid at about 18 hours, 6 hours and 1.4 hours, respectively, indicating that the nanosize significantly accelerated the hydrolysis reaction, thereby quickly producing H2. In addition, 78% of H2 was released from CSN with a particle size of 300nm within 24 hours. Therefore, for the conventional dose of one suppository per day, CSN with a particle size of 300nm is more suitable for constructing the required multi-drug suppositories to maximize the bioavailability of H2 in the colon. Subsequently, fatty acid glyceride (FAG) was used as the matrix, carbomer was used as the adhesive, 300nm CSN was used as the hydrogen-producing substance, and 5-FU was used as the chemotherapy drug, in which FAG encapsulated 5-FU and CSN to prepare 5-FU / CSN@FAG suppositories, as shown by FT-IR spectrum ( Figure 5 ) and Raman spectroscopy ( Figure 6), 5-FU / CSN@FAG suppository is a mixture of FAG, CSN, 5-FU and carbomer.

[0056] Example 2

[0057] Effect verification: 5-FU / CSN@FAG was constructed with FAG: 120 mg; 5-FU: 3 mg; CSN: 12 mg.

[0058] First, the stability of FAG and 5-FU / CSN@FAG suppositories at room temperature and their melting properties at body temperature were evaluated. Figure 7 DSC data showed that FAG was mainly composed of two glycerides with different melting points (32.4℃ and 37.5℃). When 20mg / g 5-FU and 80mg / g CSN were added to FAG, the melting point of FAG increased slightly (33.0℃ and 38.6℃), but it did not significantly hinder the gradual melting of 5-FU / CSN@FAG suppositories at 37℃ (ref. Figure 3 c, d and Figure 8 ), 5-FU / CSN@FAG suppositories can be completely melted within 30 minutes at 37°C, so that they can adhere to the intestinal wall in time and achieve sustained release of the drug. In addition, after about one year of storage, the melting behavior of the suppository did not change ( Fig. 9 ), the XRD results of CSN extracted from suppositories still maintain the crystal structure of CeSi2 ( Fig.10 ), indicating that the suppository has good stability.

[0059] In order to evaluate the distribution and retention of 5-FU / CSN@FAG suppositories in the intestine, FAG was labeled with Cy5-COOH to construct fluorescently traceable 5-FU / CSN@Cy5-FAG suppositories. After fasting for 24 hours, 5-FU / CSN@Cy5-FAG suppositories were administered rectally, and the retention and distribution behavior of the suppositories in the colorectum were monitored by fluorescence imaging. Figure 2 As shown in Figure e, the suppository had completely melted and filled the entire colon within half an hour. Within 24 hours after the suppository was administered, obvious fluorescent signals could be observed in the colorectal area, indicating that the suppository could be stably present in the colon. After 24 hours of administration, almost all of the suppositories had been discharged from the colon due to the peristalsis and defecation of the mice ( Figure 2 e and f). Therefore, the 5-FU / CSN@FAG suppository designed by the present invention can basically meet the requirement of once-daily suppository administration.

[0060] 2. Drug / H2 release behavior of suppositories in vivo and in vitro

[0061] The present invention studied the release behavior of 5-FU and H2 from 5-FU / CSN@FAG suppositories in vitro by in vitro simulation experiments. First, gas chromatography (GC) was used to measure the H2 released from CSN and 5-FU / CSN@FAG suppositories. Fig.14 As can be seen from a in FIG, encapsulating CSN into suppositories does not significantly change the hydrolysis H2 release behavior of CSN, because a sufficient amount of water can penetrate into the suppository to hydrolyze CSN. It can be found that the hydrogen production can reach 76% of the theoretical value within 24 hours, which means that once-daily administration of 5-FU / CSN@FAG suppositories can achieve great H2 bioavailability. In addition, the present invention is based on the standard curve of 5-FU ( Fig.11 ), calculate the release amount of 5-FU. Fig.14 As shown in b), the 5-FU release rate of 5-FU / CSN@FAG suppositories is slightly higher than that of 5-FU@FAG suppositories, which may be because the H2 released by CSN accelerates the diffusion of 5-FU from the suppository. Importantly, about 81% of 5-FU is continuously released from 5-FU / CSN@FAG suppositories within 24 hours, which is conducive to the sufficient bioavailability of 5-FU / CSN@FAG suppositories within 24 hours of retention in the colon.

[0062] In addition, the release of 5-FU and H2 in vivo was further measured. 5-FU was labeled with FITC to construct 5-FU-FITC / CSN@FAG suppositories, which were administered to mice with colon tumors via the rectal route. At fixed time points, major organs such as the intestine, heart, liver, spleen, lungs, and kidneys were extracted for in vitro fluorescence imaging. Fig.14 As can be seen from c and d in Figure 5, the fluorescence of 5-FU-FITC in 5-FU-FITC / CSN@FAG suppositories was severely quenched at the beginning (0h), but recovered after the suppository was released (other time points). In addition, a large amount of 5-FU-FITC filled the entire colon only 0.5h after administration. Only a small part of 5-FU-FITC diffused to the small intestine within 2-4 hours, while no 5-FU-FITC was observed in other organs such as the heart, liver, spleen, lungs, and kidneys within 24 hours, reflecting the advantages of suppositories in local drug release and avoiding the toxic side effects of drugs on the whole body.

[0063] In addition to fluorescent tracing, high performance liquid chromatography (HPLC) was used to measure the concentration of free 5-FU in the colon and small intestine. Fig.14 The results in e further confirmed that 5-FU was mainly distributed in the colon rather than in the small intestine, and its concentration in the colon gradually decreased over time, probably due to the excretion of the suppository. It is worth noting that the 5-FU concentration in the blood is quite low ( Fig.12 ), which is significantly lower than the concentration in the colon ( Fig.14e). Therefore, these low-concentration 5-FU molecules may be cleared from the mouse body in time instead of accumulating in major normal organs such as the heart, liver, spleen, lungs, and kidneys, thereby minimizing the toxic side effects of 5-FU on these important organs. On the other hand, the H2 concentration in colon tumors at different time points was measured using a hydrogen electrode. Fig.14 As can be seen from figure f, the concentration of H2 in the tumor gradually increased over time and reached 150 μM within 6 hours, which is sufficient to assist chemotherapy, thereby enhancing the anti-cancer effect and reducing side effects.

[0064] 3. In vitro efficacy and mechanism of hydrogen and chemotherapy

[0065] like Fig.15 As shown in a, the suppository was placed in a transwell chamber, and cells were seeded at the bottom of the chamber, so that 5-FU and H2 released from the suppository acted on the cells, and cytotoxicity was measured after incubation for 24 hours. Fig.15 b and c in Figure 13-15 The results showed that 5-FU suppositories showed nonspecific cytotoxicity to normal cells NCM460 cells (colon epithelial cell line) and cancer cells CT26 cells (mouse colon cancer cell line) and HCT116 cells (human colon cancer cell line), and at the same concentration (20g / g 5-FU), 5-FU had a significantly higher cytotoxicity to NCM460 cells (the cell death rate of NCM-460 was 73% ( Fig.15 b), the CT26 cell death rate was 49% ( Fig.15 c), the death rate of HCT116 cells was 56% ( Fig.13 ), reflecting that 5-FU as a conventional chemotherapy drug has significant side effects. However, CSN can significantly reduce the cytotoxicity of 5-FU to NCM460 cells ( Fig.15 b and Fig.16 ), 5-FU showed cytotoxicity to both cancer cells in a CSN content-dependent manner ( Fig.15 c in Fig.13 , Fig.17 and Fig.18 ), confirming that H2 released from CSN has anti-cancer selectivity and protects normal cells. In addition, we used NCM460 cells ( Fig.19 and Fig. 20 ), CT26 cells ( Fig.21 a and Fig. 22 ) and HCT116 cells ( Fig.21 b and Fig.23 ) proved the anticancer selectivity of CSN@FAG. When the mass ratio of CSN to 5-FU was 6:1, the anticancer enhancement and toxicity reduction effect of CSN on 5-FU was no longer obvious. Fig.15b and c in Fig.13 ), therefore, this paper selected 5-FU / CSN@FAG suppositories with a CSN / 5-FU ratio of 4:1 (80 mg / gCSN, 20 mg / g5-FU) for subsequent in vivo studies.

[0066] In order to clarify the mechanism of action of hydrogen chemotherapy, the present invention verifies that normal cells ( Fig.15 d) and cancer cells ( Fig.15 h). First, for NCM460 cells, 5-FU can cause oxidative stress and overexpression of intracellular reactive oxygen species (ROS) ( Fig.15 e and f), inhibiting NCM460 cell energy metabolism ( Fig.15 g), thereby inducing apoptosis. In contrast, H2 can effectively remove highly cytotoxic ROS ( Fig.15 e and f) and correct cellular energy metabolism ( Fig.15 g), showing an important and interesting role in protecting normal cells from 5-FU oxidative damage. On the other hand, for CT26 colon cancer cells, it is well known that 5-FU causes DNA damage in cancer cells and induces apoptosis, which was further confirmed in CT26 cells using 5-FU suppositories ( Fig.24 ). In addition, in the hypoxic microenvironment of the tumor, H2 can reduce CO2 to CO under the catalysis of iron-porphyrin in the mitochondria. The present invention further confirms that CT26 colon cancer cells can indeed produce a large amount of CO in an oxygen-deficient and H2-rich incubator ( Fig.15 i and j), high concentrations of CO inhibited mitochondrial uptake in cancer cells, as evidenced by a time-dependent decrease in ATP expression ( Fig.15 k in Fig.25 ), thereby inducing apoptosis of cancer cells. The two different pathways of 5-FU and H2 (or CSN) work synergistically to enhance the anticancer efficacy of 5-FU / CSN@FAG suppositories.

[0067] 4. The therapeutic effect of hydrogen combined with chemotherapy on colon cancer

[0068] To evaluate and monitor the in vivo therapeutic effect of 5-FU / CSN@FAG suppositories on colon cancer, we established an orthotopic CT26-LUC colon tumor model in Balb / c mice ( Fig.26 The tumors were tracked at fixed time points using bioluminescent fluorescence imaging. After 21 days, when the tumors grew to about 60 cubic centimeters, the mice were given suppositories once a day. The administration of suppositories was stopped once the tumors disappeared and resumed once the tumors recurred ( Fig.26 a). Fig.26The results in b and c showed that the mice in the control group began to die on the 18th day and died rapidly around the 25th day, which may be mainly attributed to the intestinal obstruction and prolapse caused by the rapid growth of the tumor. Fig.26 d) and mouse mortality ( Fig.26 In contrast, CSN@FAG suppository treatment could inhibit the growth of colon tumors to a certain extent and extend the life span of mice by 2.4-2.6 times ( Fig.26 c), although the tumor was not completely eliminated. In contrast, although 5-FU@FAG suppository treatment effectively controlled the overgrowth of the tumor as early as day 11, the mortality rate of mice was always higher than that of the CSN@FAG group. This was due to the toxic side effects of 5-FU and the gradual weight loss of mice ( Fig. 27 Most importantly, stop taking suppositories once the tumor has resolved ( Fig.26 The tumor will recur after 1-2 weeks and cause the mouse to die quickly. This situation of chemotherapy with severe side effects, tumor recurrence and rapid death is very common in clinical practice, but it is rarely taken seriously. Fortunately, 5-FU / CSN@FAG suppository treatment can not only eliminate colon tumors earlier (as early as the 7th day and completed on the 21st day) ( Fig.26 b and h), and no mice died during the entire 60-day treatment period ( Fig.26 c) or tumor recurrence ( Fig.26 b and h), indicating the important role of CSN in continuously releasing H2. In addition, the weight of tumor mice treated with 5-FU / CSN@FAG suppositories continued to increase throughout the treatment process, indicating that H2 has the effect of reducing the toxic side effects of chemotherapy. In addition, the mice in the control and FAG groups eventually died due to increased tumor volume or intestinal obstruction before tumor metastasis, but the colorectal cancer model we established still has the risk of metastasis. The tumors of mice in the 5-FU / CSN@FAG suppository group were completely eliminated before tumor metastasis, indicating that 5-FU / CSN@FAG suppositories have the effect of inhibiting colorectal cancer metastasis. Based on the above in vivo experimental results, after treatment with 5-FU / CSN@FAG suppositories, the tumor inhibition rate of colorectal cancer mice reached 100%, and there was no tumor recurrence for at least 53 days, and the survival rate of mice with colon tumors was 100%.

[0069] In addition, the present invention also conducted histopathological evaluation to study the effects of suppositories on normal colon and tumor tissues. Except for the 5-FU / CSN@FAG group, the remaining tumor mice were euthanized on the 60th day of treatment and their colon tissues were dissected for hematoxylin and eosin (H&E) and immunohistofluorescence staining. Fig.28As shown in a, in the control group, the crypts of the intestinal mucosa were generally damaged to a certain extent, and the density of goblet cells was greatly reduced, which may be due to the colon inflammation caused by the tumor. Both CSN@FAG and 5-FU@FAG treatments significantly improved this change in mucosal crypts and goblet cells, which should be attributed to the anti-inflammatory effect of H2 released by CSN and the anti-cancer effect of 5-FU, respectively. Fig.28 The whole colon image shown in b shows that the colon tissue in the control group was severely inflamed and the intestinal wall was obviously serrated and thickened. After treatment with 5-FU / CSN@FAG, it was completely recovered, while the other groups recovered more slowly, which was due to the dual anti-tumor and anti-inflammatory effects of 5-FU / CSN@FAG suppositories. Fig.28 As shown in Figures b and c, the size and tumor cell density of colon tumors in the CSN@FAG, 5-FU@FAG, and 5-FU / CSN@FAG groups were significantly smaller than those in the control group, which may be due to the killing effect of 5-FU and H2 on tumor cells. Immunofluorescence staining further showed that all the residual tumor cells in the 5-FU / CSN@FAG group were dead ( Fig.28 e and f), no proliferation activity ( Fig.28 d and f in the figure), while the other groups did not have this phenomenon. This well explains why the tumors in mice treated with 5-FU@FAG suppositories are prone to recurrence, while the tumors in the 5-FU / CSN@FAG group did not recur ( Fig.26 In normal colon tissue, the levels of IL-6 and TNF-α, two typical inflammatory markers commonly used to reflect inflammation, in the CSN group and 5-FU / CSN group were significantly lower than those in the other groups ( Fig.28 g, h, and i), which is consistent with the H&E results ( Fig.28 This indicates that H2 released from CSN can effectively reduce 5-FU-induced colon inflammation, which is consistent with the results of in vitro experiments ( Fig.15 ), which also explains why the tumor mice treated with 5-FU / CSN@FAG suppositories gained weight and had a 100% survival rate ( Fig.26 ).

[0070] Finally, the present invention evaluated the rectal irritation of the suppository on mice. The mice were given one tablet per day for one week, and the rectum was taken for H&E staining, and no redness, swelling, congestion, rectal prolapse, or diarrhea was observed ( Fig.29 ), H&E staining results showed ( Fig.30 ) No obvious abnormalities were found in the rectal mucosa of each group, indicating that the suppositories in each group did not irritate the mouse rectum during administration. In addition, at the end of the treatment, the mouse heart, liver, spleen, lung, kidney and tumor tissues were dissected and H&E stained for safety assessment. Fig.31 The results showed that none of the treatment groups caused significant damage to these major organs, which may be related to the relatively low concentration of 5-FU in the blood ( Fig.12 ), indicating that the developed suppositories have good biocompatibility in mice. In addition, suppositories with different contents of 5-FU and / or CSN (CSN@FAG, 5-FU@FAG and 5-FU / CSN@FAG) were administered to mice once a day, and blood samples were collected three days after the administration of suppositories to test hematological, liver, kidney and cardiac indicators. Figure 32-34 The results showed that there were no significant differences in the parameters of all these indicators between the treatment group and the control group, confirming that the local application of the developed suppository under the conditions of the present invention did not cause hematological toxicity, liver, kidney or heart damage to mice.

[0071] Example 3

[0072] The MgSi2 raw material powder was crushed into nanoparticles with an average particle size of about 200nm to 900nm, or 200-500nm, preferably 300nm by ultrasonic crushing. MgSi2 nanoparticles (MSN) have a higher hydrogen production efficiency. Subsequently, polyethylene glycol was used as a matrix, carbomer was used as a binder, 300nm MSN was used as a hydrogen-producing substance, and capecitabine (Cape) was used as a chemotherapy drug, wherein polyethylene glycol (PEG) encapsulated capecitabine and magnesium hydride nanoparticles to prepare Cape / MSN@PEG suppositories.

[0073] Effect verification: Cape / MSN@PEG was constructed with PEG: 120 mg; Cape: 3 mg; MSN: 12 mg.

[0074] 1. Drug / H2 release behavior of suppositories in vivo and in vitro

[0075] The present invention studied the release behavior of Cape and H2 from Cape / MSN@PEG suppositories in vitro by in vitro simulation experiments. First, GC was used to measure the release of H2 from MSN and Cape / MSN@PEG suppositories. Fig.35 As can be seen from a in Figure 1, encapsulating MSN into suppositories does not change its H2 release behavior and trend, indicating that water molecules can penetrate into Cape / MSN@PEG suppositories and react with MSN to generate H2. It can be found that the hydrogen production can reach 80% of the theoretical value within 12 hours, which means that applying Cape / MSN@PEG suppositories twice a day can achieve great H2 bioavailability. As shown in ( Fig.35 As shown in b), the release rate of Cape from Cape / MSN@PEG suppositories was slightly higher than that from Cape@PEG suppositories. This phenomenon may be because the H2 produced by the hydrolysis of MSN accelerated the diffusion of Cape from the suppository.

[0076] The Cape / MSN@PEG suppository was placed in a transwell chamber, and tumor / normal cells were seeded at the bottom of the chamber to allow the Cape and H2 released from the suppository to act on the cells. After incubation for 24 hours, the cell survival rate was measured. Fig.36 As shown in the results, Cape suppositories showed nonspecific cytotoxicity to NCM460 cells (normal cells), CT26 cells, and HCT116 cells (cancer cells), and at the same concentration (20 g / g Cape), Cape had a significantly higher cytotoxicity to NCM460 cells (the cell death rate of NCM-460 was 77% ( Fig.36 a), the CT26 cell death rate was 38% ( Fig.36 b), HCT116 cell death rate was 41% ( Fig.36 c), because Cape can be converted into cytotoxic 5-FU. The use of Cape / MSN@PEG suppositories can significantly reduce the cytotoxicity of Cape to NCM460 cells ( Fig.36 a), 5-FU showed cytotoxicity to both cancer cells in a CSN content-dependent manner ( Fig.36 b and c), confirming that H2 released by MSN has anti-cancer selectivity and protects normal cells.

Claims

1. A medicinal suppository for treating colorectal cancer, characterized in that: The drug suppository is composed of a chemotherapy drug, a hydrogen-producing material that slowly produces hydrogen when in contact with water, and a temperature-sensitive matrix material. The mass ratio of the chemotherapy drug, the hydrogen-producing material, and the temperature-sensitive matrix material is 1-20:5-50:50-500.

2. The medicinal suppository for treating colorectal cancer according to claim 1, characterized in that: The chemotherapy drugs include one or more of 5-fluorouracil, capecitabine, S-1, raltitrexed, oxaliplatin or irinotecan.

3. The medicinal suppository for treating colorectal cancer according to claim 1, characterized in that: The hydrogen-producing material includes one or more of CeSi2, MgH2, CaH2, CaSi2 or MgSi2 nanoparticles.

4. The medicinal suppository for treating colorectal cancer according to claim 1, characterized in that: The solid-liquid transition temperature of the thermosensitive matrix material is 34 to 38 degrees Celsius, and includes one or more of cocoa butter, semi-synthetic coconut oil ester, semi-synthetic litsea cubeba ester, semi-synthetic palm oil ester, glycerol gelatin, polyethylene glycol, poloxamer or fatty acid glyceride.

5. Use of the pharmaceutical suppository according to claim 1 in the treatment of colorectal cancer, characterized in that: When embolized into the body, the drug suppository simultaneously releases hydrogen molecules and the chemotherapy drugs it carries.

6. Use of the pharmaceutical suppository according to claim 5 in the treatment of colorectal cancer, characterized in that: The synchronously released hydrogen molecules can in situ reduce the toxic side effects of chemotherapy drugs on normal intestinal tissues, and at the same time in situ enhance the anti-cancer effect of chemotherapy drugs on colorectal cancer cells.

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