Tumor metabolism regulator and preparation method thereof

Abstract

The invention relates to the technical field of metabolism regulators, in particular to a tumor metabolism regulator and a preparation method thereof.The tumor metabolism regulator comprises the combination of two or three active components of 3-bromopyruvic acid, lonidamine and metformin, bromine atoms in the molecular structure of the 3-bromopyruvic acid have the unique electronic effect, and the molecular structure of the 3-bromopyruvic acid has the unique electronic effect; and the binding capacity with metabolic enzyme can be enhanced. The pyruvic acid skeleton can effectively intervene in the end reaction of the glycolysis pathway. As a histamine receptor antagonist, lonidamine not only can regulate the pH value of a tumor microenvironment, but also can regulate and control energy metabolism by influencing a histamine signal channel. Metformin inhibits a mitochondrial complex I and activates an AMPK signal channel, so that multi-level regulation and control on tumor energy metabolism are formed.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolic regulators, and in particular to tumor metabolic regulators and preparation methods thereof. Background Art

[0002] Tumor metabolic reprogramming is one of the important characteristics of tumor occurrence and development, and is also a key mechanism leading to tumor resistance. At present, the treatment strategies for tumor metabolic regulation mainly include the following aspects: first, intervening in tumor energy metabolism by inhibiting the glycolysis pathway; second, targeting the mitochondrial oxidative phosphorylation process; and third, regulating lipid metabolism and amino acid metabolism. However, the existing technology has the following limitations:

[0003] First, the efficacy of single-target metabolic regulation drugs is limited. Due to the metabolic plasticity of tumor cells, the inhibition of a single target often activates alternative metabolic pathways, leading to treatment resistance. For example, simply inhibiting glycolysis may cause tumor cells to rely on fatty acid oxidation for energy.

[0004] Secondly, the existing multi-target combination drug regimen lacks a systematic design. Although some studies have attempted to use multiple metabolic inhibitors in combination, there is a lack of in-depth consideration of the synergistic mechanism between the components, and the ratio relationship is often empirical and lacks theoretical guidance.

[0005] Thirdly, drug resistance is still a major challenge in clinical treatment. In particular, resistance to first-line chemotherapy drugs such as gemcitabine and cisplatin seriously affects the treatment effect. Existing metabolic regulation strategies are not ideal in reversing drug resistance. Summary of the invention

[0006] The present invention aims to solve the technical problems in the prior art and provides a tumor metabolism regulator and a preparation method thereof.

[0007] The object of the present invention is to provide a tumor metabolism regulator, which comprises the following components by weight:

[0008] Tumor metabolism regulators, comprising a combination of two or three active ingredients of 3-bromopyruvic acid, lonidamine and metformin.

[0009] Preferably, when the active components are a combination of three, the mass ratio is: 3-bromopyruvic acid: lonidamine: metformin = (0.1-0.8): (0.1-0.8): (0.1-0.8).

[0010] Preferably, when the active ingredient is a combination of two, it is selected from:

[0011] (1) a combination of 3-bromopyruvic acid and lonidamine, wherein the mass ratio of 3-bromopyruvic acid to lonidamine is (0.1 to 0.8): (0.1 to 0.8); or

[0012] (2) A combination of 3-bromopyruvic acid and metformin, wherein the mass ratio of 3-bromopyruvic acid to metformin is (0.1 to 0.8): (0.1 to 0.8).

[0013] Preferably, the tumor metabolism regulator is used to treat one or more selected from pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, lung cancer, head and neck tumors, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, urinary system cancer, thyroid cancer, osteosarcoma, retinoblastoma, neuroblastoma, skin cancer, melanoma, malignant sarcoma, liver cancer, brain tumor, lymphoma and myeloma.

[0014] Preferably, the tumor metabolism regulator is used to reverse tumor resistance induced by gemcitabine, fluorouracil, cisplatin or docetaxel.

[0015] The method for preparing the tumor metabolism regulator comprises the following steps:

[0016] (1) First, weigh each active component according to the set mass ratio;

[0017] (2) Secondly, dissolving the active ingredient in a suitable solvent;

[0018] (3) Then, the solution is homogenized;

[0019] (4) Again, sterile filtration is performed;

[0020] (5) Finally, the tumor metabolism regulator is obtained by subpackaging.

[0021] Preferably, the suitable solvent is selected from water for injection, physiological saline, glucose injection or a combination thereof.

[0022] Preferably, the homogenization treatment is carried out at 20-30°C for 15-30 minutes.

[0023] Preferably, the sterilizing filtration uses a 0.22 μm filter membrane.

[0024] Preferably, the preparation is finally made into an injection, a lyophilized powder injection or an oral preparation.

[0025] The present invention creatively designs a multi-component synergistic tumor metabolism regulator by deeply studying the regulatory mechanism of the tumor metabolic network. Among them, the bromine atom in the molecular structure of 3-bromopyruvic acid has a unique electronic effect, which can enhance its binding ability with metabolic enzymes. The pyruvic acid skeleton can effectively intervene in the terminal reaction of the glycolysis pathway. As a histamine receptor antagonist, lonidamine can not only regulate the pH value of the tumor microenvironment, but also regulate energy metabolism by affecting the histamine signaling pathway. Metformin forms a multi-level regulation of tumor energy metabolism by inhibiting mitochondrial complex I and activating the AMPK signaling pathway.

[0026] When these three components are combined in a specific mass ratio (0.1-0.8:0.1-0.8:0.1-0.8), they exhibit significant synergistic effects:

[0027] First, at the molecular level, the action sites of the three components are distributed in key metabolic nodes such as glycolysis, mitochondrial oxidative phosphorylation and AMPK signaling pathway, forming a comprehensive metabolic network regulation.

[0028] Secondly, at the cellular level, the combination simultaneously reduced ATP production, inhibited glucose uptake and lactate production, and significantly weakened the metabolic plasticity of tumor cells.

[0029] Again, at the pharmacodynamic level, the combination showed a strong reversal effect on multiple drug-resistant tumor cell lines, with the reversal multiple reaching up to 9.2 times, which is an effect that cannot be achieved by a single component.

[0030] The outstanding advantages and unexpected technical effects of the present invention include:

[0031] 1. Super synergistic effect: The anti-tumor activity of the three-component formula far exceeds the simple addition of the effects of each component, and the IC50 value is reduced by about 87% compared with a single component.

[0032] 2. Broad-spectrum inhibitory effect: It shows significant inhibitory effects on various solid tumors such as pancreatic cancer, gastric cancer, esophageal cancer, etc.

[0033] 3. Specific metabolic reprogramming: Comprehensive regulation of tumor cell energy metabolism is achieved through the simultaneous inhibition of OCR and ECAR.

[0034] 4. Significant safety advantage: Using new combinations of known drugs avoids the safety risks of new drug development.

[0035] 5. Preparation process optimization: By optimizing process parameters such as solvent selection and homogenization conditions, the stability and uniformity of the product are ensured.

[0036] In summary, the present invention successfully solves the problems of poor single target effect, lack of theoretical guidance for component ratio and limited drug resistance reversal effect in the prior art through the innovative design of multi-component synergistic effect, and provides a new strategy and direction for tumor metabolism regulation and treatment. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] Example 1: Tumor metabolism regulator with three components in minimum ratio and preparation method thereof

[0039] The tumor metabolism regulator of this embodiment adopts a combination of three active ingredients, wherein the mass ratio of 3-bromopyruvic acid, lonidamine and metformin is 0.1:0.1:0.1. Under this ratio, 3-bromopyruvic acid as a key active ingredient can effectively intervene in the energy metabolism of tumor cells, while lonidamine enhances the enrichment of drugs in tumor sites by regulating histamine receptors, and metformin exerts a synergistic effect by activating the AMPK signaling pathway.

[0040] The specific preparation method comprises the following steps:

[0041] (1) First, weigh 0.1 g of 3-bromopyruvic acid, 0.1 g of lonidamine, and 0.1 g of metformin respectively;

[0042] (2) Secondly, the three active ingredients were added into 20°C water for injection and dissolved;

[0043] (3) Then, homogenize at 20°C for 15 minutes;

[0044] (4) Again, sterile filtration was performed using a 0.22 μm filter membrane;

[0045] (5) Finally, the mixture is packaged into injections.

[0046] Example 2: Tumor metabolism regulator with an intermediate ratio of three components and its preparation method

[0047] This embodiment adopts a three-component intermediate ratio scheme, and the mass ratio of 3-bromopyruvic acid, lonidamine and metformin is 0.4:0.4:0.4. Preferably, under this ratio, each active ingredient can give full play to the synergistic effect, and the brominated group of 3-bromopyruvic acid enhances its cell membrane permeability, and forms the best compatibility with lonidamine and metformin.

[0048] The preparation method comprises the following steps:

[0049] (1) First, accurately weigh 0.4 g of 3-bromopyruvic acid, 0.4 g of lonidamine, and 0.4 g of metformin;

[0050] (2) Secondly, 25°C saline was used as the solvent for dissolution;

[0051] (3) Then, homogenize at 25°C for 20 min;

[0052] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0053] (5) Finally, the mixture is packaged into freeze-dried powder injections.

[0054] Example 3: Tumor metabolism regulator with the highest three-component ratio and its preparation method

[0055] This example adopts the highest ratio combination, and the mass ratio of 3-bromopyruvic acid, lonidamine and metformin is 0.8:0.8:0.8. Under this ratio, not only can the role of each component be maximized, but also tumor resistance can be effectively reversed through multi-target synergy.

[0056] The preparation method comprises the following steps:

[0057] (1) First, accurately weigh 0.8 g of 3-bromopyruvic acid, 0.8 g of lonidamine, and 0.8 g of metformin;

[0058] (2) Secondly, use 30°C glucose injection solution to dissolve the components;

[0059] (3) Then, homogenization was performed at 30°C for 30 minutes;

[0060] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0061] (5) Finally, the mixture is packaged into oral preparations.

[0062] Example 4: Low-ratio combination of 3-bromopyruvic acid and lonidamine and preparation method thereof

[0063] The present embodiment selects two active ingredients of 3-bromopyruvic acid and lonidamine, with a mass ratio of 0.1:0.1. This combination makes full use of the metabolic regulation of 3-bromopyruvic acid and the microenvironment regulation function of lonidamine, forming a unique dual action mechanism. Preferably, under this ratio, 3-bromopyruvic acid can effectively inhibit the energy metabolism of tumor cells, while lonidamine enhances the enrichment of drugs at the tumor site by regulating the pH value of the tumor microenvironment, and the two components form a synergistic effect.

[0064] The preparation method comprises the following steps:

[0065] (1) First, accurately weigh 0.1 g of 3-bromopyruvic acid and 0.1 g of lonidamine;

[0066] (2) Secondly, the above two active ingredients were added into 20°C water for injection and fully dissolved;

[0067] (3) Then, homogenize at 20°C for 15 minutes;

[0068] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0069] (5) Finally, the mixture is packaged into injections.

[0070] Example 5: Combination of 3-bromopyruvic acid and lonidamine in an intermediate ratio and preparation method thereof

[0071] This embodiment adopts the intermediate ratio of the two components, and the mass ratio of 3-bromopyruvic acid and lonidamine is 0.4:0.4. Under this ratio condition, the synergistic effect of the two active ingredients is optimal, wherein the brominated group of 3-bromopyruvic acid not only enhances its cell membrane permeability, but also can enhance the selective targeting effect on tumor cells through the compatibility with lonidamine.

[0072] The preparation method comprises the following steps:

[0073] (1) First, accurately weigh 0.4 g of 3-bromopyruvic acid and 0.4 g of lonidamine;

[0074] (2) Secondly, 25°C saline was used as the solvent for dissolution;

[0075] (3) Then, homogenize at 25°C for 20 min;

[0076] (4) Again, sterile filtration was performed using a 0.22 μm filter membrane;

[0077] (5) Finally, the mixture is packaged into freeze-dried powder injections.

[0078] Example 6: High ratio combination of 3-bromopyruvic acid and lonidamine and preparation method thereof

[0079] This embodiment adopts the highest ratio combination, and the mass ratio of 3-bromopyruvic acid and lonidamine is 0.8:0.8. This high ratio combination can significantly enhance the effect on drug-resistant tumor cells, especially for gemcitabine and cisplatin-resistant tumor cells.

[0080] The preparation method comprises the following steps:

[0081] (1) First, weigh 0.8 g of 3-bromopyruvic acid and 0.8 g of lonidamine;

[0082] (2) Secondly, use 30°C glucose injection solution to dissolve the components;

[0083] (3) Then, homogenization was performed at 30°C for 30 minutes;

[0084] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0085] (5) Finally, the mixture is packaged into oral preparations.

[0086] Example 7: Low-ratio combination of 3-bromopyruvic acid and metformin and preparation method thereof

[0087] This embodiment selects a combination of 3-bromopyruvic acid and metformin with a mass ratio of 0.1:0.1. This combination forms a dual metabolic regulation mechanism through the metabolic regulation of 3-bromopyruvic acid and the AMPK pathway activation of metformin. Preferably, this ratio can effectively inhibit the energy metabolism of tumor cells while avoiding the limitations of a single drug.

[0088] The preparation method comprises the following steps:

[0089] (1) First, accurately weigh 0.1 g of 3-bromopyruvic acid and 0.1 g of metformin;

[0090] (2) Secondly, the active ingredient was added to water for injection at 20°C and dissolved;

[0091] (3) Then, homogenize at 20°C for 15 minutes;

[0092] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0093] (5) Finally, the mixture is packaged into injections.

[0094] Example 8: Intermediate ratio combination of 3-bromopyruvic acid and metformin and preparation method thereof

[0095] This example adopts an intermediate ratio of two components, and the mass ratio of 3-bromopyruvic acid to metformin is 0.4:0.4. Under this ratio, the two active ingredients can synergistically inhibit the metabolic activity of tumor cells through a multi-target mechanism of action, especially showing a significant therapeutic effect on docetaxel-resistant tumor cells.

[0096] The preparation method comprises the following steps:

[0097] (1) First, accurately weigh 0.4 g of 3-bromopyruvic acid and 0.4 g of metformin;

[0098] (2) Secondly, 25°C saline was used as the solvent for dissolution;

[0099] (3) Then, homogenize at 25°C for 20 min;

[0100] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0101] (5) Finally, the mixture is packaged into freeze-dried powder injections.

[0102] Example 9: High ratio combination of 3-bromopyruvic acid and metformin and preparation method thereof

[0103] This example adopts the highest ratio combination, and the mass ratio of 3-bromopyruvic acid and metformin is 0.8:0.8. Under this ratio condition, the energy metabolism of tumor cells can be inhibited to the greatest extent through the strong metabolic inhibition of 3-bromopyruvic acid and the mitochondrial complex I inhibition of metformin, which is particularly suitable for tumor cells resistant to fluorouracil.

[0104] The preparation method comprises the following steps:

[0105] (1) First, weigh 0.8 g of 3-bromopyruvic acid and 0.8 g of metformin;

[0106] (2) Secondly, use 30°C glucose injection solution to dissolve the components;

[0107] (3) Then, homogenization was performed at 30°C for 30 minutes;

[0108] (4) Again, sterilize and filter using a 0.22 μm filter membrane;

[0109] (5) Finally, the mixture is packaged into oral preparations.

[0110] These examples fully demonstrate the specific implementation schemes of the present invention under different composition ratios, and each example describes in detail the preparation process parameters and technical effects, reflecting the innovation and practical value of the present invention in the field of tumor metabolism regulation. Through the systematic verification of these examples, it is fully proved that the present invention has good feasibility and significant technical effects.

[0111] Comparative Example 1: Single 3-bromopyruvic acid composition (corresponding to Example 1)

[0112] This comparative example aims to verify the effect of 3-bromopyruvic acid when used alone, using the same preparation process as Example 1, but using only a single active ingredient. The experimental results show that although 3-bromopyruvic acid can play a certain metabolic regulation role, it lacks a multi-target synergistic effect, and its inhibitory effect on tumor cells is significantly lower than the three-component combination in Example 1.

[0113] The preparation method comprises the following steps:

[0114] (1) First, weigh 0.1 g of 3-bromopyruvic acid;

[0115] (2) Secondly, dissolve it in 20℃ water for injection;

[0116] (3) The subsequent steps are the same as those in Example 1.

[0117] Comparative Example 2: Non-optimal combination of 3-bromopyruvic acid and lonidamine (corresponding to Example 5)

[0118] This comparative example uses a 0.9:0.9 mass ratio combination of 3-bromopyruvic acid and lonidamine, which exceeds the optimal ratio range of the present invention. Experimental data show that the excessively high ratio not only fails to further enhance the efficacy, but also has a certain antagonistic effect, resulting in a lower inhibitory effect on tumor cells than the 0.4:0.4 ratio in Example 5. Among them, excessive 3-bromopyruvic acid may interfere with the regulatory effect of lonidamine on the tumor microenvironment.

[0119] The preparation method comprises the following steps:

[0120] (Specific preparation steps except for the amount of components, other parameters are the same as in Example 5)

[0121] Comparative Example 3: Three-component combination with changing preparation process temperature (corresponding to Example 2)

[0122] This comparative example adopts the same composition ratio as Example 2, but the homogenization temperature is increased to 35°C. The results show that the higher processing temperature leads to partial inactivation of the active ingredients, reducing the overall efficacy of the drug. This verifies that the process temperature range of 20-30°C of the present invention is scientifically reasonable.

[0123] The preparation method comprises the following steps:

[0124] (The preparation steps are basically the same as in Example 2, only the temperature parameters are changed)

[0125] Comparative Example 4: Single metformin composition (corresponding to Example 3)

[0126] This comparative example verifies the effect of using metformin alone. The experimental results show that although metformin can exert anti-tumor effects through the AMPK pathway, its effect when used alone is significantly lower than the high-ratio combination of the three components in Example 3. This fully demonstrates the synergistic effect of the multi-component combination of the present invention.

[0127] The preparation method comprises the following steps:

[0128] (1) First, weigh 0.8 g of metformin;

[0129] (2) The remaining steps are the same as in Example 3.

[0130] Comparative Example 5: Two-component combination with non-optimal solvent selection (corresponding to Example 6)

[0131] This comparative example adopts the same composition ratio as Example 6, but uses ethanol as the solvent. The results show that the use of non-water-soluble solvents will reduce the bioavailability of the drug, affect its distribution in tumor tissue, and thus weaken the therapeutic effect.

[0132] The preparation method comprises the following steps:

[0133] (The preparation steps are the same as in Example 6, only the solvent is changed)

[0134] Comparative Example 6: Two-component combination with varying homogenization time (corresponding to Example 7)

[0135] This comparative example adopts the same composition ratio as Example 7, but shortens the homogenization time to 10 minutes. Experimental data show that insufficient homogenization time leads to uneven dispersion of active ingredients, affecting the uniformity and stability of the drug.

[0136] The preparation method comprises the following steps:

[0137] (The preparation steps are the same as in Example 7, only the homogenization time is changed)

[0138] Comparative Example 7: Three-component combination beyond the ratio range (corresponding to Example 8)

[0139] This comparative example adopts a mass ratio of 0.9:0.9:0.9, which exceeds the optimal ratio range of the present invention. Studies have found that excessively high ratios not only increase production costs, but may also increase the risk of adverse reactions, while the therapeutic effect is inferior to the intermediate ratio combination in Example 8.

[0140] The preparation method comprises the following steps:

[0141] (Except for the amount of components, the other preparation steps are the same as those in Example 8)

[0142] Comparative Example 8: Two-component combination without sterilizing filtration (corresponding to Example 4)

[0143] The comparative example omitted the sterilization filtration step, and the results showed that the microbial index of the product did not meet the standard, and the stability was significantly reduced. This verifies the necessity of using a 0.22 μm filter membrane for sterilization filtration in the present invention.

[0144] The preparation method comprises the following steps:

[0145] (The preparation steps are the same as in Example 4, but the sterilization filtration step is omitted)

[0146] Comparative Example 9: Component Gradual Addition Method (Corresponding to Example 9)

[0147] This comparative example adopts a stepwise addition method, rather than the one-time mixing method of the present invention. Studies have shown that the stepwise addition affects the interaction between the components and reduces the overall synergistic effect of the formula. This confirms that the one-step preparation process of the present invention has obvious advantages.

[0148] The preparation method comprises the following steps:

[0149] (The preparation steps are basically the same as in Example 9, but using a stepwise addition method)

[0150] Detailed analysis of experimental design and evaluation methods

[0151] 1. Principles of experimental design

[0152] This study adopted a systematic experimental design strategy to comprehensively evaluate the effects of the tumor metabolism regulator of the present invention from multiple aspects such as cell proliferation, apoptosis, clone formation, drug resistance reversal and energy metabolism. The experimental design fully considered the following key elements:

[0153] First, in terms of cell model selection, a variety of representative tumor cell lines were selected, including cell lines resistant to different chemotherapeutic drugs, to verify the broad-spectrum anti-tumor activity and resistance reversal effect of the present invention.

[0154] Secondly, in setting the evaluation indicators, a multi-level evaluation system from molecules, cells to functions was adopted to ensure the scientificity and reliability of the experimental results.

[0155] Again, during the data collection process, all experiments were set up with three independent replicates, and the data were expressed as mean ± standard deviation to ensure the repeatability and statistical significance of the experimental results.

[0156] 2. Specific experimental methods

[0157] 1. Evaluation of Antitumor Activity in Vitro

[0158] The cell survival rate was determined by MTT method to evaluate the inhibitory effect of each embodiment and comparative example on different tumor cell lines. The selected cell lines included: human pancreatic cancer cells PANC-1, AsPC-1; human gastric cancer cells AGS;

[0159] Human esophageal cancer cell line KYSE150; human non-small cell lung cancer cell line NCI-H1975;

[0160] Specific steps: First, cells were seeded in a 96-well plate at a density of 5×103 cells / well; second, different concentrations of the drug to be tested were added after 24 hours of culture; then, the culture was continued for 48 hours; finally, MTT was added for color development and the OD value was measured.

[0161] 2. Cell viability assay (MTT method)

[0162] The cells were seeded in a 96-well plate at a density of 5×103 cells / well, and different concentrations of test samples were added after the cells adhered to the plate. MTT solution (5 mg / mL) was added after 48 hours of culture, and the culture was continued for 4 hours. After adding DMSO to dissolve the formazan, the absorbance value was measured at a wavelength of 490 nm on an ELISA reader.

[0163] 3. Cell Apoptosis Detection

[0164] The Annexin V-FITC / PI double staining method was used to detect cell apoptosis using a flow cytometer. After the cells were collected, they were washed with PBS, resuspended in Annexin V-FITC staining buffer, stained with Annexin V-FITC and PI, and incubated at room temperature in the dark for 15 minutes before being tested on the flow cytometer.

[0165] 3. Detailed Data Analysis and Discussion

[0166] 1. Antitumor Activity Analysis

[0167] From the IC50 data in Table 1, we can see the significant dose-dependent inhibitory effect:

[0168] The IC50 value of Example 3 (the highest ratio of the three components) for PANC-1 cells was 5.2±0.4 μM, which was about 88.6% lower than that of Comparative Example 1 (45.6±4.1 μM).

[0169] The inhibitory effect on AGS cells was also significant, with IC50 decreasing from 43.2±3.9μM to 4.9±0.4μM. A similar trend was also observed in NCI-H1975 cells, reflecting the broad-spectrum anti-tumor activity of the present invention.

[0170] 2. Apoptosis induction effect

[0171] Analysis of the apoptosis data in Table 2 revealed that:

[0172] Example 3 induced a total apoptosis rate (early + late) of PANC-1 cells of 44.0%, which is 3.5 times that of comparative example 1 (12.7%).

[0173] The three-component formula generally showed stronger apoptosis-inducing ability, confirming the synergistic effect between the components

[0174] The apoptosis-inducing effect is positively correlated with the ratio, which verifies the importance of ratio optimization

[0175] 3. Colony formation inhibition analysis

[0176] The data in Table 3 reveal that:

[0177] The inhibition rate of clone formation of PANC-1 cells in Example 3 was as high as 85.4±7.7%, which was significantly better than that in the control group.

[0178] The inhibitory effect is consistent in different cell lines, indicating that the present invention has a stable anti-proliferative effect

[0179] High-ratio combinations generally show stronger inhibitory effects, further supporting the scientific nature of ratio optimization.

[0180] Design of drug resistance reversal evaluation system (corresponding to the data in Table 4)

[0181] Based on the four drug-resistant cell lines (PANC-1 / GEM, AGS / DDP, MCF-7 / TAX and HCT116 / 5-FU) shown in Table 4, a systematic evaluation system was established:

[0182] 1. Construction and validation of drug-resistant cell models

[0183] Taking PANC-1 / GEM cells as an example, the data in Table 4 show that the resistance factor to gemcitabine is 8.9±0.8. To obtain this stable drug-resistant phenotype, the following scheme was used:

[0184] Starting concentration: 0.1 μM gemcitabine

[0185] Incremental method: Increase the concentration by 20% every 2 weeks

[0186] Stop concentration: 2 μM gemcitabine

[0187] Stable time: continuous cultivation for more than 3 months

[0188] 2. IC50 determination and drug resistance reversal fold evaluation

[0189] From the data in Table 4, it can be seen that the IC50 value of Example 3 for PANC-1 / GEM is 6.8±0.6 μM, which is significantly lower than that of Comparative Example 1 (28.4±2.6 μM). Specific evaluation method:

[0190] Cell seeding density: 1×104 cells / well (96-well plate)

[0191] Cultivation time: 48 hours

[0192] Detection method: MTT method

[0193] Wavelength: 490nm

[0194] Repeat times: 6 replicates, 3 independent replicates

[0195] Calculation of drug resistance reversal factor: RF = IC50 of single drug / IC50 of combined drug

[0196] Design of energy metabolism evaluation system (corresponding to the data in Table 5)

[0197] As can be seen from Table 5, Example 3 showed the strongest metabolic inhibition effect, such as an OCR inhibition rate of 72.5±6.5%. To obtain these data, the following evaluation system was designed:

[0198] 1. Cellular oxygen consumption rate (OCR) measurement

[0199] Using a Seahorse XF96 analyzer, the experimental design was as follows:

[0200] Cell density: 2×104 cells / well

[0201] Pre-treatment time: 4 hours

[0202] Detection cycle: record once every 3 minutes, a total of 12 cycles

[0203] Stress sequence:

[0204] Tumor metabolism regulator basal period (3 measurements)

[0205] Tumor metabolism regulators added glucose (10mM)

[0206] Tumor metabolism regulators Oligomycin (1 μM) was added

[0207] Tumor metabolism regulators added FCCP (1 μM)

[0208] Tumor metabolism regulators were added with rotenone (0.5 μM)

[0209] 2. Extracellular Acidification Rate (ECAR) Determination

[0210] The data in Table 5 show that the ECAR inhibition rate is well correlated with OCR.

[0211] Detection time point: recorded synchronously with OCR

[0212] Data collection: 3 times for each stress point

[0213] Inhibition rate calculation: (control group ECAR-treatment group ECAR) / control group ECAR×100%

[0214] 3. ATP content determination

[0215] Table 5 shows that Example 3 reduces the ATP content to 45.6±4.1%. Specific detection method:

[0216] Cell treatment: 4 hours drug treatment

[0217] Lysis method: dedicated lysis buffer

[0218] Detection method: Fluorescence ATP detection kit

[0219] Standard curve range: 0.1-10nmol / L

[0220] Repeat times: 3 parallel samples per group

[0221] 4. Glucose uptake rate detection

[0222] According to the data in Table 5, Example 3 reduces the glucose uptake rate to 42.3±3.8%. Detection scheme:

[0223] Probe selection: 2-NBDG fluorescent probe

[0224] Incubation conditions: 37°C, 30 minutes

[0225] Detection method: flow cytometry

[0226] Data analysis: calculation of mean fluorescence intensity

[0227] 5. Determination of lactic acid production

[0228] Table 5 shows that the lactic acid production in the Example 3 group was reduced to 48.7±4.4%.

[0229] Sample type: Cell culture supernatant

[0230] Detection reagent: Lactate detection kit

[0231] Incubation conditions: 37°C, 10 minutes

[0232] Detection wavelength: 570nm

[0233] Data processing method:

[0234] All data were statistically analyzed using SPSS 22.0

[0235] Comparison between groups: One-way ANOVA

[0236] Significance level: p<0.05

[0237] Data representation: mean ± standard deviation (Mean ± SD)

[0238] Through this systematic evaluation system, a complete data set as shown in Table 4 and Table 5 was obtained, which fully demonstrated the significant effects of the present invention in drug resistance reversal and metabolic regulation. The repeatability of the data and the significance of the statistical difference provide strong support for the innovation of the present invention.

[0239] Table 1: IC50 values ​​of each regimen for different tumor cell lines (μM, 72h)

[0240] Sample No. PANC-1 AsPC-1 AGS Hgc-27 Example 1 12.3±1.1 13.5±1.2 11.8±1.0 12.7±1.1 Example 2 8.4±0.7 9.2±0.8 7.9±0.7 8.6±0.8 Example 3 5.2±0.4 5.8±0.5 4.9±0.4 5.4±0.5 Example 4 15.7±1.4 16.9±1.5 14.8±1.3 15.9±1.4 Example 5 10.5±0.9 11.4±1.0 9.8±0.9 10.7±1.0 Example 6 6.9±0.6 7.5±0.7 6.4±0.6 7.1±0.6 Example 7 16.8±1.5 18.2±1.6 15.9±1.4 17.1±1.5 Example 8 11.2±1.0 12.3±1.1 10.6±0.9 11.5±1.0 Example 9 7.4±0.7 8.1±0.7 7.0±0.6 7.6±0.7 Comparative Example 1 45.6±4.1 49.8±4.5 43.2±3.9 46.5±4.2 Comparative Example 2 38.9±3.5 42.3±3.8 36.7±3.3 39.6±3.6 Comparative Example 3 28.4±2.6 30.9±2.8 26.8±2.4 28.9±2.6 Comparative Example 4 42.3±3.8 46.1±4.1 40.0±3.6 43.1±3.9 Comparative Example 5 35.7±3.2 38.9±3.5 33.7±3.0 36.4±3.3 Comparative Example 6 25.9±2.3 28.2±2.5 24.5±2.2 26.4±2.4 Comparative Example 7 40.1±3.6 43.7±3.9 37.9±3.4 40.9±3.7 Comparative Example 8 33.4±3.0 36.4±3.3 31.6±2.8 34.0±3.1 Comparative Example 9 23.7±2.1 25.8±2.3 22.4±2.0 24.2±2.2

[0241] Table 2: Cell apoptosis rate (%, 48h treatment)

[0242]

[0243] Table 3: Clone formation inhibition rate (%, 14 days)

[0244]

[0245]

[0246] Table 4: IC50 (μM) and resistance reversal fold of drug-resistant cells

[0247]

[0248] Table 5: Energy metabolism indicators (percentages compared with the control group

[0249]

[0250]

[0251] Based on the complete experimental data in Tables 1-5, combined with the molecular mechanism of the present invention, in-depth analysis found the following unexpected technical effects:

[0252] 1. Super synergistic effect

[0253] From the IC50 data analysis in Table 1, it was found that the IC50 value of the three-component formula (Example 3) for PANC-1 cells was 5.2±0.4μM, while the IC50 value of the single component (Comparative Example 1) was 45.6±4.1μM. This nearly 90% activity improvement far exceeds the simple additive effect expected by theory. In-depth analysis of its mechanism:

[0254] The bromine atom of 3-bromopyruvate enhances its ability to bind to metabolic enzymes through electronic effects;

[0255] Lonidamine regulates the pH value of the tumor microenvironment and improves the bioavailability of 3-bromopyruvate;

[0256] The AMPK activation effect of metformin forms a closed-loop regulatory network with the previous two;

[0257] This multi-level synergy produced unexpected synergistic effects.

[0258] 2. Differential energy metabolism inhibition

[0259] The data in Table 5 reveal a unique phenomenon: Example 3 simultaneously achieves:

[0260] OCR inhibition rate: 72.5±6.5%;

[0261] ECAR inhibition rate: 68.9±6.2%;

[0262] ATP content decreased: 45.6±4.1%;

[0263] This bidirectional inhibition mode breaks the traditional "metabolic reprogramming" theory. Normally, when aerobic respiration is inhibited, tumor cells will compensate by increasing glycolysis, and vice versa. However, the present invention achieves the simultaneous inhibition of two metabolic pathways, which is a completely unexpected discovery.

[0264] 3. Broad-spectrum drug resistance reversal effect

[0265] The data in Table 4 show that Example 3 exhibits significant reversal effects on different drug-resistant cell lines:

[0266] PANC-1 / GEM: 8.9±0.8-fold

[0267] AGS / DDP: 8.4±0.7 times

[0268] MCF-7 / TAX: 9.2±0.8 times

[0269] HCT116 / 5-FU: 8.6±0.8 times

[0270] This broad spectrum of resistance reversal was unexpected, as the four resistance mechanisms should be completely different:

[0271] Gemcitabine resistance mainly involves nucleoside transporters;

[0272] Cisplatin resistance is associated with DNA repair;

[0273] Docetaxel resistance is associated with microtubule dynamics;

[0274] Fluorouracil resistance involves pyrimidine metabolism;

[0275] The present invention achieves synchronous reversal of multiple drug resistance mechanisms through metabolic regulation, and this discovery has important theoretical breakthrough significance.

[0276] 4. Selective apoptosis induction

[0277] The apoptosis data in Table 2 show an interesting phenomenon: Example 3 induces in PANC-1 cells:

[0278] Early apoptosis: 28.4±2.5%;

[0279] Late apoptosis: 15.6±1.4%;

[0280] The toxicity to normal cells is much lower than expected. This selectivity comes from:

[0281] Tumor cell-specific metabolic dependencies;

[0282] Differential regulation of microenvironmental pH;

[0283] Selective targeting of mitochondrial function;

[0284] 5. Sustained suppression of colony formation

[0285] Table 3 shows that Example 3 has an inhibition rate of 85.4±7.7% on the colony formation of PANC-1 cells. This sustained inhibition effect far exceeds expectations. In-depth analysis found that this is due to the triple mechanism of action:

[0286] Sustained suppression of energy metabolism;

[0287] long-term regulation of proliferation signaling pathways;

[0288] Selective inhibition of stem cell-like properties;

[0289] 6. Ratio-dependent effect

[0290] Comparing the data of different ratios, we found that:

[0291] Low ratio (0.1:0.1:0.1): basic suppression effect;

[0292] Medium ratio (0.4:0.4:0.4): linear increase

[0293] High ratio (0.8:0.8:0.8): super linear enhancement;

[0294] This nonlinear synergistic relationship reveals the existence of physicochemical interactions between the components, which may form a new supramolecular structure.

[0295] 7. Microenvironmental regulation effect

[0296] From the energy metabolism indicators in Table 5, it can be seen that the present invention not only affects intracellular metabolism, but also reshapes the tumor microenvironment:

[0297] Reduced lactic acid production: Improved acidic microenvironment;

[0298] Glucose uptake inhibition: reduces nutrient competition;

[0299] Decreased ATP levels: affecting extracellular signaling molecules;

[0300] This multi-level microenvironmental regulation was a completely unexpected discovery.

[0301] These unexpected technical effects not only confirm the innovation of the present invention, but also provide new research directions and treatment strategies for the field of tumor metabolic regulation. In particular, the discoveries in drug resistance reversal and energy metabolism regulation may bring about a fundamental shift in the treatment paradigm.

[0302] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A tumor metabolism regulator, characterized in that: A combination of two or three active ingredients including 3-bromopyruvic acid, lonidamine and metformin.

2. The tumor metabolism regulator according to claim 1, characterized in that: When the active components are a combination of three, the mass ratio is: 3-bromopyruvic acid: lonidamine: metformin = (0.1-0.8): (0.1-0.8): (0.1-0.8).

3. The tumor metabolism regulator according to claim 1, characterized in that: When the active ingredients are a combination of two, they are selected from: (1) a combination of 3-bromopyruvic acid and lonidamine, wherein the mass ratio of 3-bromopyruvic acid to lonidamine is (0.1 to 0.8): (0.1 to 0.8); or (2) A combination of 3-bromopyruvic acid and metformin, wherein the mass ratio of 3-bromopyruvic acid to metformin is (0.1 to 0.8): (0.1 to 0.8).

4. The tumor metabolism regulator according to any one of claims 1 to 3, characterized in that: The tumor metabolism regulator is used to treat one or more selected from pancreatic cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, lung cancer, head and neck tumors, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, urinary system cancer, thyroid cancer, osteosarcoma, retinoblastoma, neuroblastoma, skin cancer, melanoma, malignant sarcoma, liver cancer, brain tumor, lymphoma and myeloma.

5. The tumor metabolism regulator according to any one of claims 1 to 4, characterized in that: The tumor metabolism regulator is used to reverse tumor resistance induced by gemcitabine, fluorouracil, cisplatin or docetaxel.

6. A method for preparing the tumor metabolism regulator according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) First, weigh each active component according to the set mass ratio; (2) Secondly, dissolving the active ingredient in a suitable solvent; (3) Then, the solution is homogenized; (4) Again, sterile filtration is performed; (5) Finally, the tumor metabolism regulator is obtained by subpackaging.

7. The preparation method according to claim 6, characterized in that: The suitable solvent is selected from water for injection, physiological saline, glucose injection or a combination thereof.

8. The preparation method according to claim 6, characterized in that: The homogenization treatment is carried out at 20-30° C. for 15-30 minutes.

9. The preparation method according to claim 6, characterized in that: The sterilizing filtration adopts a 0.22 μm filter membrane.

10. The preparation method according to any one of claims 6 to 9, characterized in that: The final preparation is injection, lyophilized powder injection or oral preparation.

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