Application of combination of bufalin and heme in preparation of cancer treatment medicine

Abstract

The invention relates to the technical field of medicines, and discloses an application of combination of bufalin and heme in preparation of a cancer treatment medicine. According to the invention, by means of an active metabonomics means, a key active metabolite-heme is locked through exogenous library screening and endogenous analysis, and the drug effect of bufalin is regulated by means of the active metabolite. Through verification, it is found that heme can cooperate with the synergistic drug effect in various cancer cell lines, and a new technical normal form based on an active metabolome for improving the drug effect of bufalin is provided. The pharmaceutical composition can improve the apoptosis induction ability of bufalin to different cancer cells, and provides powerful guiding significance for developing a novel treatment scheme in clinical medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of bufalin and hematin in the preparation of cancer treatment drugs. BACKGROUND

[0002] Active metabolomics focuses on exploring the functional role of metabolites at the metabolome scale. By revealing the dynamic changes of metabolic networks and their role in different biological states, active metabolites that play a key role in disease progression and drug treatment are found. By targeting key active metabolites, the phenotype is adjusted, the key pathways of disease development are revealed, and potential therapeutic targets are ultimately mined.

[0003] Cancer is one of the major diseases that the world is concerned about, especially lung cancer, liver cancer and other cancers, with high incidence and refractory, has become the focus of research by experts and scholars. The current main treatment methods include organ transplantation, tumor resection, chemotherapy, radiotherapy and targeted therapy, etc.

[0004] Traditional Chinese medicine, as an important medical means of Chinese heritage for thousands of years, as early as in the "Rites of Zhou" described "ulcer doctors are responsible for the treatment of swelling, ulcer, gold ulcer, and ulcer, and the treatment of qi, kill the Ziqi" records the method of traditional Chinese medicine for cancer treatment, and the classic works such as "Shennong's Herbal Classic" and "Compendium of Materia Medica" have made a comprehensive record and arrangement of anti-tumor traditional Chinese medicine. In recent years, based on the modern medical and research system, experts and scholars have gradually clarified the active ingredients and main mechanisms hidden under complex traditional Chinese medicine prescriptions with the help of modern pharmacological techniques. Among them, traditional Chinese medicine toad venom, as the dried secretion of Bufo gargarizans or Bufo melanostictus, has the effects of detoxification, pain relief, opening the orifice and refreshing the spirit, and is used to treat sore throat, heatstroke, coma, abscess, carbuncle, and other diseases. In recent years, there have been numerous studies on traditional Chinese medicine toad venom, which has shown potential for treating cancer, cardiovascular disease, inflammation, etc. Its corresponding Chinese medicine, Huachan Suo soft capsule, as a broad-spectrum anti-tumor drug for the digestive system, has been used in clinical practice. Bufalin, as the key active ingredient of toad venom, has shown great potential in cancer treatment through research.

[0005] Current literature suggests that bufalin exerts anti-cancer activity through various mechanisms such as promoting autophagy and apoptosis. It is particularly effective in refractory and drug-resistant cancers, reducing the risk of treatment failure by reducing drug resistance, making it a very promising anti-cancer drug. However, despite its great potential as an anti-cancer drug, bufalin has not been able to be translated to the clinic due to its narrow therapeutic window, high toxicity, and poor water solubility.

[0006] The prior art mainly improves the efficacy of bufalin through structural modification, dosage form optimization and the like, CN103083239A discloses a kind of bufalin liposome, which is composed of liposome bilayer and bufalin, wherein the liposome bilayer contains phospholipid, sterol and PEG modifier. The bufalin liposome can be prepared by film method, injection method or freeze-thaw method, has stronger anticancer effect and lower toxic side effect than bufalin monomer drug, and can be used for the treatment of cancer including liver cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, esophageal cancer, gastric cancer or leukemia.

[0007] Currently, it is urgent to create a new paradigm to show another possibility of drug synergy, CN10723335A discloses the application of drug combination in the preparation of antibacterial drugs and antibacterial drugs. The drug contains bufalin, ester bufadienolide and farwa bufadienolide; the weight ratio of bufalin, ester bufadienolide and farwa bufadienolide is 1-3:1-2:1-2. The drug is a new composition of antibacterial drugs, wherein bufalin, ester bufadienolide and farwa bufadienolide can exert synergistic antibacterial effect after mixing, especially when the weight ratio of bufalin, ester bufadienolide and farwa bufadienolide is 1.5-2.5:1-1.8:1, the synergistic antibacterial effect is more obvious; in addition, bufalin, ester bufadienolide and farwa bufadienolide can have mutual attenuation effect after mixing. However, there is no report on the synergistic study of bufalin based on active metabolomics and effective synergistic combination. SUMMARY

[0008] The present application aims to solve the problem of insufficient synergistic study of bufalin in the prior art, and provides the application of bufalin and hematin in the preparation of cancer treatment drugs. The drug combination can improve the apoptosis-inducing ability of bufalin on different cancer cells, and provides strong guidance for the development of new treatment programs in clinical medicine.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: The application of bufalin and hematin in the preparation of cancer treatment drugs.

[0010] By means of active metabolomics, the present application locks the key active metabolites through exogenous library screening and endogenous analysis, and adjusts the efficacy of bufalin by means of active metabolites. It is found that hematin can synergistically improve the efficacy of bufalin in various cancer cell lines, and provides a new technical paradigm based on active metabolomics for improving the efficacy of bufalin.

[0011] The cancer includes one or more of cervical cancer, lung cancer, prostate cancer, breast cancer and liver cancer.

[0012] The drug can induce apoptosis of cancer cells.

[0013] The heme in the medicine can improve the apoptosis-inducing ability of bufalin on cancer cells.

[0014] The administration molar concentration of the heme in the medicine is 500nM-30μM.

[0015] The administration molar concentration of the bufalin in the medicine is 1μM or less.

[0016] The medicine further comprises a pharmaceutically acceptable carrier, which is any one or a combination of a binder, a disintegrant, a lubricant, and a glidant.

[0017] The application further provides a medicine for cancer treatment, wherein the main active components of the medicine comprise bufalin and heme; the medicine achieves the treatment purpose by inducing apoptosis of cancer cells.

[0018] The medicine is used for treating one or more of cervical cancer, lung cancer, prostate cancer, breast cancer, and liver cancer.

[0019] The medicine is in any one of a liquid injection, a powder injection, a tablet, a capsule, a powder, a pill, an oral liquid, a plaster, a granule, and a dressing.

[0020] Compared with the prior art, the application has the following beneficial effects: The application finds a key active metabolite-heme based on metabolic library screening, improves the efficacy of bufalin through metabolite regulation, and has better anti-cancer efficacy than single drug and certain extensive applicability. 1. Stronger targeting: Directly regulates endogenous metabolic pathways, utilizes existing biological transformation mechanisms to enhance efficacy, and avoids off-target effects caused by exogenous drugs.

[0021] 2. Higher safety: Relies on the natural metabolic pathways of organisms, reduces the cumulative risk of toxicity caused by structural modification or combination drug use.

[0022] 3. Shorter development cycle: Based on known metabolite regulation networks, some preclinical screening stages can be skipped to accelerate the research and development process.

[0023] 4. Significant cost-effectiveness: Avoids complex dosage form processes or compatibility studies of combination drugs, and reduces production costs.

[0024] 5. Adapt to individual differences: Through metabolic regulation, the patient's metabolic phenotype differences can be flexibly adapted, and the potential of precision treatment is improved. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure is the metabolic library screening result in Example 1.

[0026] Figure 2 For the administration of hemin screened in Example 1 to increase the killing effect of bufalin on cells, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and all comparisons are compared with the non-hemin group (bufalin single drug group) at the same bufalin concentration.

[0027] Figure 3 Based on the metabonomics analysis of bufalin administration, (A) is PCA analysis, (B) is pathway enrichment analysis, and (C) is the change in the level of metabolites detected in the glutathione metabolism pathway and the porphyrin metabolism pathway.

[0028] Figure 4 For the synergistic effect of hemin and bufalin in different cell lines.

[0029] Figure 5 For the administration of hemin in Example 4 to further promote the generation of reactive oxygen species (ROS) induced by bufalin.

[0030] Figure 6 For the administration of hemin in Example 5 to inhibit the tumor growth curve of mice in combination with bufalin.

[0031] Figure 7 For the body weight change curve of mice in Example 5. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the following will further describe the present application in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. Those skilled in the art can modify or replace equivalently on the basis of understanding the technical scheme of the present application, without departing from the spirit and scope of the technical scheme of the present application, and all should be covered within the protection scope of the present application.

[0033] The raw materials used in the following specific embodiments are all purchased from the market, and the experimental methods not specified in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0034] Example 1 In the present application, the metabolic library screening method is used to obtain active metabolites that improve the killing effect of bufalin. Human cervical cancer cell line: Hela cells are used as experimental objects, and the metabolic library selected is the screening library of MCE, which contains 480 kinds of metabolites.

[0035] 1. Plating: When HeLa cells reached 80% confluence, they were trypsinized, centrifuged (900 rpm, 4 min), resuspended in DMEM medium, and counted under a microscope using a cell counting chamber. The cell concentration was diluted to 4.0 × 10⁻⁶. 4 Cell suspensions of 100 μL / well were plated in 96-well plates and incubated at 37°C with 5% CO2 for 12 h.

[0036] 2. Grouping and Drug Treatment: The experimental groups were divided into a normal group, a model group, and a drug treatment group. The normal group received no drug treatment; the model group received bufotalin IC50 treatment; and the drug treatment groups received 10 μM and 100 μM metabolite concentrations, respectively, in addition to the treatment given to the model group. After 12 hours of cell adhesion, the old solution was discarded, and the cells were treated with drugs for 24 hours according to their respective groups.

[0037] 3. Cell viability assay: Cell viability was determined using the CCK-8 assay. The sample solution in the 96-well plate was discarded, and basal medium containing 10% CCK-8 reagent was added. The plate was incubated for 1-2 hours, and the absorbance was measured at 450 nm.

[0038] 4. Data Processing: Export the absorbance results obtained from the test and save them in Excel. Then calculate cell viability using the following formula: Cell Viability (%) = (OD) Drug -OD Blank ) / (OD Control -OD Blank )×100.

[0039] OD Drug OD values ​​for the drug-treated group and the model group; OD Blank OD values ​​for cell-free blank wells (containing only culture medium and CCK-8, with the drug-treated groups containing additional drug solution); OD Control This represents the OD value of the normal group.

[0040] 5. The metabolites obtained from the initial screening were re-screened to identify stable metabolites with synergistic effects. After determining the effective and non-cytotoxic concentrations of the metabolites, 10 gradient concentrations of bufotalin (starting at 1000 μM, 2:5 dilution to 3.90625 μM, with a 0 concentration control) were set up to detect the synergistic effects of the metabolites at different bufotalin concentrations.

[0041] 6. Results are as follows Figure 1 and Figure 2As shown in the figure. By screening a metabolic library containing 480 metabolites, it was found that some metabolites could enhance the cytotoxic effect of bufotoxin. Among them, hemin and its upstream metabolite coproporphyrin III could stably exert a synergistic effect with bufotoxin. Further screening revealed that the concentrations of hemin with synergistic effect but no cytotoxicity were 1.25 μM, 2.5 μM, and 5 μM. Figure 2 The heme selected in Example 1 was used to enhance the cell-killing effect of bufotalin. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. All comparisons were made compared to the non-hemin group (bufotalin monotherapy group) at the same bufotalin concentration. At a fixed heme concentration, gradient administration of bufotalin was performed, and it was found that exogenous administration of heme increased the cell-killing effect of bufotalin.

[0042] Example 2: To further investigate the potential link between bioactive metabolites and bufotoxin based on metabolomics analysis, a cell line: human cervical cancer cell line: HeLa cells was used as the experimental subject for mass spectrometry analysis.

[0043] 1. Plating: When HeLa cells reached 80% confluence, they were trypsinized, centrifuged (900 rpm, 4 min), resuspended in DMEM medium, and counted under a microscope using a cell counting chamber. The cell concentration was diluted to 1.4 × 10⁻⁶. 5 Cell suspensions of 1.5 mL / well were plated into 6-well plates and incubated at 37°C with 5% CO2 for 12 hours.

[0044] 2. Drug treatment: The experiment was divided into a normal group and a drug treatment group. The normal group received no drug treatment; the drug treatment groups were treated with low concentration (10 nM) and high concentration (20 nM) of bufotoxin, respectively. After 12 hours of cell adhesion, the old solution was discarded, and the cells were treated with the drug for 24 hours according to their respective groups.

[0045] 3. Sample Pretreatment: Discard the sample solution and wash 2-3 times with pre-cooled PBS. Add extraction buffer, scrape and collect the cell suspension in an EP tube using a cell scraper. Repeat the freeze-thaw cycle in liquid nitrogen 3 times, incubate at -20℃ for 1 hour, and then centrifuge (4℃, 14000 rpm) for 20 minutes. Transfer the supernatant to a liquid chromatography vial for subsequent instrument analysis.

[0046] 4. Mass Spectrometry Analysis: High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS) was used for detection in this experiment. The specific detection method is as follows: An ACQUITY UPLC HSS T3 column (2.1×100mm, 1.8μm) was selected as the liquid chromatography system, the column temperature was 40℃, the injection volume was 5μL, the flow rate was 0.3mL / min, the mobile phase A was 0.03% formic acid aqueous solution, and the mobile phase B was 0.03% formic acid acetonitrile solution. The gradient was 0–12min, 3–100% B; 12–17min, 100% B; 17–18min, 100–3% B; 18–25min, 3% B.

[0047] 5. Data Processing: Mass spectrometry data were analyzed using R language, and graphs were plotted using software such as GraphPad Prism.

[0048] 6. Results: such as Figure 3 As shown. Figure 3 The results in A indicate that principal component analysis (PCA) can significantly distinguish the differences in metabolic profiles between the normal group and the drug-treated group. Figure 3 Enrichment analysis of the B pathway revealed significant metabolic changes in the glutathione and porphyrin metabolic pathways compared to the normal group. Both pathways are closely related to heme, suggesting that heme plays a crucial role in the mechanism of action of bufotalin. Bufotalin administration can dose-dependently alter the activity of the glutathione and porphyrin metabolic pathways. We further observed the trends in specific metabolite changes within these two pathways. like Figure 3 In the figure, C indicates P < 0.05, and ** indicates P < 0.01. All comparisons are compared to the normal group (CT). With increasing bufotoxin concentration, significant changes occurred in the levels of key metabolites in the porphyrin metabolic pathway (decreased glutamate, increased 5-aminolevulinic acid) and the glutathione metabolic pathway (decreased glutathione, decreased spermine), suggesting that bufotoxin may cause intracellular redox imbalance by interfering with these two pathways. Given that glutathione metabolism regulates heme homeostasis, and porphyrin metabolism directly participates in heme synthesis, this result further suggests the crucial role of heme in the mechanism of action of bufotoxin, elucidating the possibility that heme enhances the efficacy of bufotoxin by correcting metabolic imbalances and increasing oxidative damage.

[0049] Example 3 To investigate the synergistic effect of bufotoxin and heme in different cell lines, the following cell lines were used in the experiment: HeLa cells (human cervical cancer cells), A549 and H23 cells (non-small cell lung cancer cells), huh7 cells (human liver cancer cells), DU145 and PC3 cells (human prostate cancer cells), and MDA-MB-231 and MCF7 cells (human breast cancer cells).

[0050] 1. Plating: When cells reach 80% confluence, trypsin digestion and centrifugation (900 rpm, 4 min), resuspend in DMEM medium, dilute to a cell suspension, and plate into 96-well plates (100 μL / well). Incubate at 37°C, 5% CO2 for 12 h. The seeding quantities for each cell type are as follows: A549 cells 8000 / well; H23 cells 8000 / well; huh7 cells 6000 / well; DU145 cells 8000 / well; PC3 cells 7000 / well; MDA-MB-231 cells 6000 / well; MCF7 cells 6000 / well.

[0051] 2. Group settings: Heme dosage screening: Ten gradient concentrations of heme were used (starting at 100 μM, 2-diluted to 0.391 μM, with a 0 concentration control). Single-drug group (heme only) and combination drug group (heme + bufotoxin IC50 concentration) were set up to evaluate cell activity and toxicity and determine the effective and non-cytotoxic heme dosage.

[0052] Synergistic effect comparison: Based on the screened heme concentration, a bufotalin monotherapy group and a combination therapy group (fixed heme concentration) were set up, and 10 gradient concentrations of bufotalin were administered (starting from 1000 μM, 2:10 dilution to 3.90625 μM, with a 0 concentration control) to further analyze the synergistic effect and dose dependence.

[0053] 3. Drug treatment: After the cells adhered for 12 hours, they were treated with drugs according to the group settings and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0054] 4. Cell viability assay: Cell viability was determined using the CCK-8 assay. The sample solution in the 96-well plate was discarded, and basal medium containing 10% CCK-8 reagent was added. The plate was incubated for 1-2 hours, and the absorbance was measured at 450 nm.

[0055] 5. Data Processing: Export the results and save them in Excel. Calculate cell viability using the formula: CellViability(%) = (OD) / (CellViability(%)) Drug -OD Blank ) / (OD Control -OD Blank )×100. Where OD Drug OD values ​​for the drug-treated group; OD Blank OD values ​​for cell-free blank wells (containing only culture medium and CCK-8, with the drug-treated groups containing additional drug solution); OD Control The OD value is the value of a 0 concentration without drug treatment.

[0056] 6. Results are as followsFigure 4 As shown, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. All comparisons are compared to the single-drug group at the same concentration of bufotalin. Except for huh7 cells, which showed no statistical difference, exogenous administration of heme in other cell lines showed that it could increase the killing effect of bufotalin, indicating that this synergistic combination has broad applicability.

[0057] Example 4 To further investigate the effect of heme on apoptosis in A549 cells when combined with bufotalin, the human non-small cell lung cancer cell line A549 cells were used as the experimental subjects.

[0058] Previous experimental results demonstrated that the synergistic effect of this drug combination (heme and bufotalin) also exists in other cancer cell lines. Given that HeLa cells are a female-specific disease, our subsequent experiments will convert the cell line into human lung cancer A549 cells for further investigation. Related studies have shown that heme can promote intracellular Fe production by upregulating the activity of heme oxygenase-1 (HO-1, encoded by the HMOX1 gene). 2+ The accumulation of these substances enhances the generation of reactive oxygen species (ROS), ultimately inducing ferroptosis. Therefore, we used intracellular ROS detection experiments to further elucidate the synergistic superficial mechanism.

[0059] 1. Plating and Drug Administration: The cells were divided into four groups: normal control group, heme monotherapy group (25 μM), bufotalin monotherapy group (15 nM), and combined drug administration group. A549 cells were plated in 12-well plates; after 12 h of cell adhesion, the cells were treated with the drugs for 24 h according to their respective groups.

[0060] 2. Intracellular Reactive Oxygen Species (ROS) Detection Assay: This experiment used the DCFH-DA fluorescence staining method. After discarding the sample solution, the cells were washed 1-2 times with PBS, and then basal medium containing 10 μM DCFH-DA was added. The cells were incubated in a cell culture incubator at 37℃ and 5% CO2 for 30 min in the dark. Subsequently, the medium was replaced with Hoechst 33342 (1-5 μg / mL) and incubated for 10 min for nuclear staining. After washing 2-3 times with PBS, the cells were immediately detected under a fluorescence microscope: DCF (ROS, 488 / 525 nm) and Hoechst (nuclear, 350 / 461 nm). The entire experiment was conducted in the dark to ensure data reliability.

[0061] 3. ROS test results are as follows Figure 5 As shown, heme significantly enhances the intracellular reactive oxygen species (ROS) production induced by bufotalin, suggesting that this synergistic combination of drugs exerts its anticancer activity by synergistically activating the oxidative stress pathway.

[0062] Example 5: Evaluation of the therapeutic effect of heme combined with bufotalin on A549 non-small cell lung cancer tumor-bearing mice. Human A549 non-small cell lung cancer cell line was subcutaneously injected into nude BALB / c mice. Male nude mice, weighing 20±2g, were housed in a 12-hour light-12-hour dark environment at a constant temperature of 25℃ with free access to food. Mice were subcutaneously inoculated with 5×10⁻⁶ cells under sterile conditions. 6 A549 cell suspensions were randomly divided into 4 groups, with 7 mice in each group. After tumor formation, tumor volume was measured in mice, reaching a size of 200 mm. 3 When the diameter is 7-8 mm, administer medication.

[0063] Each group was intraperitoneally injected every two days with the carrier solvent (control group), bufotalin (1 mg / kg), heme (5 mg / kg), and a combination therapy group with the same dose. The survival of mice was observed and recorded over 16 days, and tumor growth curves and changes in mouse body weight were calculated. Tumor volume was defined as length * width * width * 0.5. Data were statistically analyzed using t-tests or ANOVA; P < 0.05 indicated a significant difference between the two groups. Animal care and usage procedures were approved by the Ethics Committee of Zhejiang University and complied with all applicable institutional and government regulations concerning the ethical use of animals (ZJU20250605).

[0064] The results are as follows Figures 6-7 As shown, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. The combination therapy group showed a more significant tumor growth inhibition effect compared with the single therapy group, and the inhibition of tumor growth was not a result of overall toxicity, because the body weight of mice remained stable during the treatment period (no statistically significant difference in body weight compared with the control group, P>0.05), indicating that the treatment regimen has good safety characteristics.

Claims

1. Application of the combined use of bufotoxin and heme in the preparation of cancer treatment drugs.

2. The application according to claim 1, characterized in that, The cancers mentioned include one or more of the following: cervical cancer, lung cancer, prostate cancer, breast cancer, and liver cancer.

3. According to claim 1, the drug can induce apoptosis in cancer cells.

4. According to claim 1, the heme in the drug can enhance the apoptosis-inducing ability of bufotalin on cancer cells.

5. In the application according to claim 1, the molar concentration of heme in the drug is 500 nM to 30 μM.

6. In the application according to claim 1, the molar concentration of bufotalin in the drug is below 1 μM.

7. The application according to claim 1, wherein the drug further comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is any one or a combination of several of a binder, a disintegrant, a lubricant, and a flow aid.

8. A drug for cancer treatment, characterized in that, The main active components of the drug include bufotalin and heme; the drug achieves its therapeutic purpose by inducing apoptosis in cancer cells.

9. The medicament according to claim 8, characterized in that, The types of cancers for which the drug is used to treat include one or more of cervical cancer, lung cancer, prostate cancer, breast cancer, and liver cancer.

10. The medicament according to claim 8, characterized in that, The drug is any one of the following forms: liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, ointment, granule or dressing.

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