Elesclomol and application of pharmaceutical composition of Elesclomol in preparation of drugs for preventing cancers

Abstract

The invention relates to the field of medicines, in particular to an application of Elesclomol and a pharmaceutical composition thereof in preparation of medicines for preventing cancers. According to the invention, Elesclomol is used, or Elesclomol and metformin are combined, so that the incidence rate of tumors can be obviously reduced, and the dryness of tumor cells can be inhibited; in animal experiments, the occurrence probability of tumors is remarkably reduced through the medicine combination and the administration mode, occurrence and development of the tumors are remarkably inhibited, and the obvious tumor prevention effect is achieved. The combined medicine is suitable for various tumors. According to the medicine combined treatment scheme, the obvious synergistic effect can be generated, the curative effect is obvious, the use is convenient and safe, the tumor occurrence probability is greatly reduced, the medical cost is reduced, and the clinical application potential is very high.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the application of Elesclomol (Iresmy) and its pharmaceutical composition for preparing a drug for preventing cancer. BACKGROUND

[0002] Cancer, as a chronic disease caused by multiple factors, seriously threatens human health. According to statistics, there are more than 3.5 million new cancer cases and more than 2 million deaths in China every year, causing a social and economic burden of more than 10 billion yuan, and the incidence of major cancers such as liver cancer and lung cancer is on the rise. Cancer, as the second leading cause of death worldwide, its prevention and control rely on systematic three-level prevention strategies. Although primary prevention (such as smoking control and vaccination) can reduce the risk of 30-40% of diseases, the prevention of drug intervention still faces significant bottlenecks. For example, tamoxifen can reduce the risk of breast cancer by 50%, but it increases the risk of thrombosis and endometrial cancer; aspirin reduces the risk of colorectal cancer by 20-40%, but it is prone to bleeding; and the effect of nutritional supplements (such as vitamin D) in preventing cancer lacks high-quality evidence support. Existing cancer prevention drugs are either narrow in scope or have significant side effects or have unclear mechanisms, so it is urgent to develop cancer prevention drugs based on new mechanisms, covering a wide range of populations, and having safety, to fill the current technical gap. SUMMARY

[0003] In view of the above research situation, the present application finds a new medical use of Elesclomol (Iresmy) through the study of the metabolic pathway of tumors.

[0004] The application of Elesclomol and its pharmaceutically acceptable salt for preparing a drug for preventing cancer.

[0005] Among them, the pharmaceutically acceptable salt of Elesclomol is sodium salt, potassium salt, triethylamine salt or meglumine salt.

[0006] The present application also provides the combined application of Elesclomol and metformin, which provides a new combined treatment method for tumor prevention of high-risk population of cancer.

[0007] The application of Elesclomol or its pharmaceutically acceptable salt, and metformin or its pharmaceutically acceptable salt, for preparing a drug for preventing cancer.

[0008] Among them, the pharmaceutically acceptable salt of Elesclomol is sodium salt, potassium salt, triethylamine salt or meglumine salt; the pharmaceutically acceptable salt of metformin is hydrochloride, maleate, lactate, acetate, cyclohexylcarboxylate, nitrate, sulfate and phosphate.

[0009] The cancer includes liver cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, leukemia, lung cancer, stomach cancer and other cancers.

[0010] The medicine is for preventing tumor for high-risk population.

[0011] The high-risk population of tumor refers to DNA repair gene mutation carriers, including BRCA1 / 2 mutation, Lynch syndrome, TP53 mutation; genetic tumor syndrome, including APC gene mutation, PTEN mutation; chronic infectious diseases, including HBV / HCV, HPV, Helicobacter pylori; precancerous lesion state, including Barrett's esophagus, cirrhosis, breast atypical hyperplasia, various irregular nodules; ionizing radiation exposure; chemical carcinogens exposure; long-term smokers; alcoholics; type 2 diabetes; abnormal tumor marker test. Among them, long-term smokers are > 30 packs per year, alcoholics are > 50g alcohol / day; obesity is BMI > 30.

[0012] The administration scheme of the combination is continuous administration of Elesclomol and pulse administration of metformin, wherein the administration frequency of metformin is not less than once a week.

[0013] In addition, the application also provides a pharmaceutical composition for preventing cancer.

[0014] A pharmaceutical composition for preventing cancer, the effective component of which is composed of a first component and a second component, The first component is Elesclomol (Iresist) or a pharmaceutically acceptable salt thereof; The second component is metformin or a pharmaceutically acceptable salt thereof.

[0015] Further, the pharmaceutically acceptable salt of the first component Elesclomol is sodium salt, potassium salt, triethylamine salt or meglumine salt; the pharmaceutically acceptable salt of the second component metformin includes hydrochloride, maleate, lactate, acetate, cyclohexylcarboxylate, nitrate, sulfate and phosphate.

[0016] Further, the pharmaceutical composition further includes pharmaceutical excipients.

[0017] Further, the pharmaceutical composition further includes various sustained-release preparations, which are matrix-type sustained release, membrane-controlled sustained release, multi-unit microparticles, liposome-encapsulated sustained release, nanoparticle sustained release.

[0018] Preferably, Elesclomol is a tablet; metformin is a hydrochloric acid sustained-release tablet.

[0019] The administration mode of the pharmaceutical composition is continuous administration of Elesclomol and pulse administration of metformin.

[0020] Further, the Elesclomol is administered orally at a dose of 2-100 mg / kg / day; and the metformin is administered at a single dose of 10-1000 mg / kg. Preferably, the Elesclomol is administered at a dose of 10 mg / kg / day, and the metformin is administered at a single dose of 100 mg / kg.

[0021] Further, the metformin is administered in pulses at a frequency of 1-5 times per week. Preferably, the metformin is administered in pulses twice per week.

[0022] Further, the metformin is administered in pulses within 2 months after the Elesclomol is administered. Preferably, the metformin is administered in pulses on the 20th day after the Elesclomol is administered.

[0023] In a third aspect, the present application provides a kit for preventing cancer, which comprises two components: Elesclomol and metformin. Further, each kit contains 7 portions of Elesclomol, each portion having a dose of 100-5000 mg, preferably 500 mg; and 1-2 portions of metformin, each portion having a dose of 500-50000 mg, preferably 5000 mg.

[0024] Further, the Elesclomol and the metformin are packaged in the kit independently or after being mixed with pharmaceutically acceptable excipients. Preferably, the Elesclomol and the metformin are packaged in the kit independently. Further, the Elesclomol and the metformin are packaged in the kit according to a time schedule. The administration schedule comprises continuous administration of the Elesclomol and pulse administration of the metformin, wherein the metformin is administered at a frequency of not less than once per week.

[0025] In the present application, Elesclomol (Irofulven) (CAS No. 488832-69-5) has the chemical structure as shown in Formula 1; and metformin (CAS No. 657-24-9) has the chemical structure as shown in Formula 2.

[0026]

[0027] Metformin, as the most widely used first-line oral drug for type 2 diabetes, has been used by more than 150 million patients since its launch in 1957. It activates the AMPK pathway to inhibit hepatic gluconeogenesis and enhance peripheral insulin sensitivity, achieving safe glycemic control. In addition to its classic hypoglycemic effect, it has been shown to have potential anti-tumor mechanisms in recent years. Its core effects include: inhibiting mTOR phosphorylation to inhibit the carcinogenic mTOR pathway, down-regulating the pro-cancer factor insulin / IGF-1 level, and interfering with mitochondrial complex I to induce cancer cell energy crisis; enhancing the antigen-presenting ability of dendritic cells and activating the killing function of CD8+ T cells, clinical studies have shown that it can reverse the resistance of non-small cell lung cancer to PD-1 inhibitors; remodeling the intestinal flora to increase the production of short-chain fatty acids (such as butyric acid), strengthening the intestinal barrier and reducing the absorption of carcinogens. In the field of tumor prevention, some studies have shown that it can reduce the risk of multiple cancers to some extent: the incidence of colorectal cancer is reduced by 37% (RR=0.63), the risk of liver cancer is reduced by 57% (animal model), the risk of prostate cancer death is reduced by 24% every 6 months of medication, and the risk of hematological tumors is reduced by 13%, but overall its monotherapy is affected by tumor type and has limited effect.

[0028] Elesclomol is a small molecule copper ionophore that selectively kills tumor cells by inducing cuproptosis. However, its clinical trials have encountered major setbacks: when combined with paclitaxel for the treatment of metastatic melanoma, it did not significantly prolong progression-free survival (PFS) and only showed limited benefits for a subgroup with low serum lactate dehydrogenase (LDH) levels.

[0029] Although the ultimate goal of tumor treatment and tumor prevention is to reduce the incidence and mortality of tumors, their focus is significantly different. Prevention focuses on reducing tumor occurrence, while treatment focuses on eliminating existing tumors and preventing recurrence and metastasis. In our tumor-related experiments, the inventors found that when Elesclomol was used as a tumor prevention drug in tumor prevention experiments, the incidence of liver cancer was greatly reduced during liver cancer formation, especially in high-ammonia combined with DEN-induced liver cancer, which was much higher than the prevention effect of the existing targeted therapy drug, lenvatinib. However, if Elesclomol is used as a treatment drug after liver cancer formation, its therapeutic effect is limited, and it is not as effective as lenvatinib. Lenvatinib, although one of the commonly used targeted tumor treatment drugs, has limited preventive effect on tumors. These experiments not only indicate that tumor treatment and tumor prevention are actually two different situations, but also indicate that a therapeutic drug cannot be directly used for prevention. Elesclomol has obvious preventive effect when used as a preventive drug, and metformin combined with Elesclomol can achieve better preventive effect, and metformin is preferably administered in a pulse manner.

[0030] The use of Elesclomol or the combination of Elesclomol and metformin in the present application can significantly reduce the incidence of tumors and inhibit the stemness of tumor cells; in animal experiments, the drug combination and administration mode significantly reduce the probability of tumor occurrence and significantly inhibit the occurrence and development of tumors, thereby achieving an obvious tumor prevention effect. The combined drug is suitable for various tumors. There is no research on the effect of "Elesclomol and metformin for preventing tumor occurrence" at home and abroad. The drug combination treatment scheme can produce obvious synergistic effect, has obvious curative effect, is convenient and safe to use, greatly reduces the probability of tumor occurrence and thereby reduces medical expenses, and has high clinical application potential.

[0031] The present application breaks through the following findings:

[0032] In the DEN-induced rat liver cancer model, the following unexpected observations are made: Prevention window effect: the incidence of tumors is reduced to 11.2% (P<0.001) when Elesclomol is administered before the formation of liver cancer; and the incidence of tumors is only reduced to 48.6% when the preventive administration of lenvatinib is advanced.

[0033] The effect of Elesclomol is significantly lower than that of lenvatinib (tumor incidence 69.2% vs 54.2%) when the drug is administered after the formation of liver cancer; Synergistic prevention: the incidence of tumors is reduced to 9.3% when metformin is synergistically administered; the incidence of tumors is reduced to 4.67% when Elesclomol and metformin are pulsed administered.

[0034] Mechanism research:

[0035] (1) The mechanism of Elesclomol: tumor stemness (i.e., the acquisition and maintenance of tumor stem cell properties) is the core engine driving tumorigenesis - it confers tumor stem cells (CSCs) with self-renewal, differentiation potential, and drug resistance through microenvironment regulation (such as hypoxia, immune cell interaction), epigenetic reprogramming (such as miRNA / DNA methylation), and cellular plasticity transformation, thereby initiating tumors and driving their malignant progression. In this process, the abnormal metabolism of cells, i.e., the reductive carboxylation process of the reverse tricarboxylic acid cycle (rTCA), drives the stemness of tumor cells, and inhibiting the abnormal metabolism of cells can block the malignant transformation of cells from the source. In our previous study, we found that rTCA exists in eukaryotic cells through metabolic flux analysis of eukaryotic cells, and FDXR and FDX1 proteins are key rate-limiting proteins in this process. Elesclomol (Iresmyl) is a copper ion carrier in previous studies, which exerts an anti-tumor effect by inducing oxidative stress and copper death, but in early clinical trials for metastatic melanoma, leukemia, and other indications, it did not meet expectations. Our study found that Elesclomol can bind to FDXR, inhibit the key protein form, and inhibit the rTCA of tumor cells, thereby playing a good role in inhibiting tumor stemness within a certain period of time, and ultimately achieving a good inhibitory effect on tumor generation (inhibition rate of about 89%).

[0036] Compared with known Elesclomol studies, the action protein, principle and pathway of Elesclomol in this patent are completely different; this patent does not involve copper death, and we excluded the possible influence of copper death in the experiment; finally we confirmed that Elesclomol only has a preventive effect, and the therapeutic effect is very limited; while the control drug lenvatinib can have a therapeutic effect, the preventive effect is very limited, which shows that prevention is not equal to treatment, and therapeutic drugs cannot be directly used for prevention, and the effectiveness of treatment is not equal to the effectiveness of prevention. That is, "the prior art is only used for the treatment of cancer and has not been thought to be used for the prevention of cancer."

[0037] (2) The mechanism of metformin: Although the use of elesclomol can better prevent the occurrence of tumors for a period of time, tumor prevention is a long-term process. A small amount of escaped tumor cells under high ammonia conditions can activate the mTOR pathway, thereby jointly causing tumors to appear after elesclomol administration. Metformin (preferably pulsed administration) can effectively inhibit the mTOR pathway and glucose uptake, promote the conversion of more glucose into lactic acid, limit rTCA, and thus play a more stable and efficient synergistic role in inhibiting tumor occurrence (inhibition rate > 95%) on a longer time scale, making it more suitable for long-term tumor prevention in high-risk tumor populations. The combination of elesclomol and metformin is suitable for the prevention and administration of various cancers. In related cell biology experiments and animal experiments, the drug components have shown a significant synergistic effect in tumor prevention, with significant efficacy, ease of use, safety, and a greatly reduced likelihood of tumor occurrence, and have great potential for clinical use. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 : M+3 of a-KG in FDX1-KD and FDXR-KD cell lines under high ammonia conditions.

[0039] Figure 2 : Stemness of FDX1-KD and FDXR-KD cell lines under high ammonia conditions.

[0040] Figure 3 : WB verification of FDX1-KD and FDXR-KD.

[0041] Figure 4 : Binding of FDX1, FDXR, and elesclomol to molecular simulation.

[0042] Figure 5 : Apoptosis rates of HepG2 and other various cells before and after the addition of elesclomol under high ammonia conditions, as well as apoptosis rates caused by different administration methods of elesclomol + metformin.

[0043] Figure 6 : M+3 of a-KG in HepG2 cells before and after the addition of elesclomol and the use of metformin under high ammonia conditions.

[0044] Figure 7 : Stemness rates of HepG2 cells before and after the addition of elesclomol and the use of metformin under high ammonia conditions.

[0045] Figure 8 : Apoptosis rates of HepG2 cells before and after the use of elesclomol after the addition of a chelating agent under high ammonia conditions.

[0046] Figure 9 : M+3 of a-KG of HepG2 cells before and after using Elesclomol with chelator first and then high ammonia.

[0047] Figure 10 : Lactate level of cells before and after using Elesclomol, Metformin and Elesclomol+Metformin pulse in high ammonia.

[0048] Figure 11 : LDH level of cells before and after using Elesclomol, Metformin and Elesclomol+Metformin pulse in high ammonia.

[0049] Figure 12 : mTOR pathway phosphorylation level of cells before and after using Elesclomol, Metformin and Elesclomol+Metformin pulse in high ammonia.

[0050] Figure 13 : Apoptosis rate of HepG2 cells before and after using Metformin.

[0051] Figure 14 : Apoptosis rate of HepG2 cells before and after using Elesclomol with other drugs in high ammonia.

[0052] Figure 15 : Tumorigenic rate in liver cancer animal model using different administration methods.

[0053] Figure 16 : HE staining of liver after treating liver cancer animal with different administration methods. DETAILED DESCRIPTION

[0054] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here.

[0055] The present application provides a pharmaceutical composition, which is essentially a drug combination for preventing tumor occurrence in high-risk tumor population, as well as a unique administration method and a kit. In order to make the patent easier to understand, some terms are defined first. As used in this application, unless otherwise explicitly defined herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0056] The reverse tricarboxylic acid cycle (rTCA) is a reverse metabolic pathway that fixes and reduces CO2, and relies on ferredoxin / NADPH to provide reducing power, and is a key adaptive mechanism for tumors to maintain biosynthesis and redox homeostasis under stress conditions. The results of the present application show that Elesclomol can inhibit cell stemness by inhibiting key proteins of rTCA, thereby preventing tumor occurrence.

[0057] Elesclomol is also known as Iresist or Iresist (CAS No: 488832-69-5).

[0058] Pharmaceutical composition: refers to a mixture containing a therapeutically effective amount of one or more of the compounds described herein and its pharmaceutically acceptable tautomer, solvate, hydrate or salt, and other pharmaceutically acceptable carriers. The purpose of preparing the compound into a pharmaceutical composition is to facilitate the administration to the treatment subject. According to the first aspect of the present application, the present application provides a pharmaceutical composition comprising a first component and a second component, wherein the first component is Elesclomol, and the second component is metformin.

[0059] According to some embodiments of the present application, the administration dose of the first component and the second component is 10 mg / kg / day; the single dose of metformin is 200 mg / kg.

[0060] According to some embodiments of the present application, the pharmaceutical composition is used for preventing tumor (cancer) occurrence.

[0061] According to some embodiments of the present application, the cancer includes but is not limited to liver cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, leukemia, lung cancer and gastric cancer, and various types of cancer.

[0062] The pharmaceutical composition of the present application comprises a safe and effective amount of the drug (active ingredient) of the present application and a pharmacologically acceptable excipient or carrier. Among them, "safe and effective amount" refers to: the amount of active ingredients is sufficient to produce a significant preventive effect without serious side effects. Generally, the pharmaceutical composition contains 0.001-1000 mg of active ingredients per dose, preferably 0.05-300 mg of active ingredients per dose, more preferably 0.5-200 mg of active ingredients per dose.

[0063] According to some embodiments of the present application, the total content of the first component and the second component is 1-100%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the pharmaceutical composition.

[0064] Drug synergy: The combination of two drugs exhibits a significant synergistic therapeutic effect: When the efficacy of the first component of a single drug is 90% and the second component of a single drug is 15%, based on the Bliss independence model (which assumes that the mechanisms of action of the drugs are independent of each other), the expected combined efficacy should be 91.5%, but the actual observed value exceeds 96%, which is significantly higher than the expected value (Δ> 4.5%), and is statistically verified (p < 0.05), which can be clearly determined as a synergistic effect; this synergy not only reflects the mathematical model, but also has clinical substantive value - it breaks through the "ceiling" of 90% efficacy of the first component of a single drug with high efficacy, and its mechanism may be due to the complementary effects of the second component of the drug and the first component of the drug in inhibiting tumor proliferation and stemness pathways, thereby enhancing the overall tumor prevention ability, providing a new strategy to overcome the bottleneck of single drug use.

[0065] The term "effective dose" refers to the amount of a drug that can achieve the prevention purpose in the body of a subject. Those skilled in the art will understand that the "effective dose" can vary depending on the administration route of the drug, the drug adjuvant used, the administration time, the physical condition of the subject, the type of tumor to be prevented, and the situation of combined use with other drugs.

[0066] The administration of the composition of the present application can be performed in various modes depending on the purpose and conditions of administration. The administration mode includes oral, injection (intravenous, intramuscular, subcutaneous), rectal, implantation. It can be prepared into any one of the pharmaceutically allowable dosage forms, including but not limited to tablets, oral agents, powders, injections, liposomes, capsules, granules, powders, suppositories, powders, solutions, etc.

[0067] The composition of the present application can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0068] According to certain embodiments of the present application, the kit further comprises one or more additional therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises a device for the corresponding administration mode, such as but not limited to a syringe.

[0069] Unless otherwise indicated in the present application or clearly contradicted by context, the terms "a", "an", "the", "at least one", and similar referents in the context of describing the present application (including the claims) are to be interpreted as covering both the singular and the plural. Unless otherwise indicated in the present application or clearly contradicted by context, the terms "comprising", "having", "including" and "containing" used in the present application are to be construed as open-ended terms (i.e., "including, but not limited to"). Unless otherwise indicated in the present application or clearly contradicted by context, all methods described in the present application can be performed in any suitable order according to the understanding of those skilled in the art.

[0070] The following are several specific examples of the present application, but it is worth noting that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments made by ordinary professionals in the field according to the content of the above application still fall within the scope of protection of the present application.

[0071] Materials and methods: Cell culture: Human cancer cell lines of different germ layers HepG2, 9810, 97H, SW13 and A549 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cell lines were grown in a 37℃, 5% CO2 environment using DMEM high glucose medium containing 10% fetal bovine serum. The production of lentivirus for stable cell line construction was completed by Hanbio Science and Technology (Shanghai, China) and used according to the manufacturer's instructions.

[0072] CCK-8 method was used to detect the apoptosis rate of cells: Cells were seeded into 96-well plates at a density of 5,000 cells per well in 100 μL of serum-containing medium and pre-cultured for 3-24 hours at 37 °C in a 5% CO2 incubator to allow cell attachment. At the time of detection, 10 μL of CCK-8 reagent was added to each well along with 90 μL of serum-free medium, and the mixture was incubated in the dark for 1-4 hours. Finally, the absorbance (OD value) was measured at 450 nm using a microplate reader, and the relative apoptosis rate was calculated according to the formula: apoptosis rate (%) = (OD of experimental group - OD of zero-adjustment well) / (OD of control group - OD of zero-adjustment well) x 100%. 450 - OD of zero-adjustment well 450 - OD of zero-adjustment well 450 - OD of zero-adjustment well 450

[0073] Isotope detection: Cells (4 x 10 13 / cm2) were cultured in medium containing 6 C-labeled glucose for 16 hours under different experimental conditions. After the medium was discarded and the residual medium was washed with normal saline, 3 mL of mass spectrometry organic solvent (methanol: acetonitrile: water = 5:3:2) was added, and the mixture was allowed to react for 15 minutes. The liquid was then removed, and the mixture was centrifuged at 12,000 rpm for 15 minutes at low temperature. The supernatant was then frozen at -80 °C for 4 hours. The freeze-dried product was re-dissolved in 200 μL of mass spectrometry organic solvent (methanol: acetonitrile: water = 5:3:2), and the mixture was centrifuged at 12,000 rpm for 15 minutes at low temperature. The supernatant was then prepared for mass spectrometry sample preparation. The QTRAP 6500 triple quadrupole linear ion trap mass spectrometer (AB Sciex, Macclesfield, UK) was used to detect the isotope content in the cells.

[0074] LDH detection: About 1 x 10 6 cells were added to 300 μL of normal saline or PBS for mechanical homogenization or ultrasonic disruption. After homogenization, the mixture was centrifuged at 10,000 g for 10 minutes at 4 °C, and the supernatant was placed on ice for detection. LDH catalyzes the production of lactic acid from pyruvic acid, and pyruvic acid reacts with 2,4-dinitrophenylhydrazine to form pyruvic acid dinitrophenylhydrazone, which is brown-red in an alkaline solution. The color intensity is directly proportional to the concentration of pyruvic acid. By measuring the OD value, the activity of LDH can be calculated.

[0075] Lactic acid detection: 5 x 10 6 cells were added to 200 μL of normal saline or PBS for homogenization. After homogenization, the mixture was centrifuged at 10,000 g for 10 minutes at 4 °C, and the supernatant was placed on ice for detection.

[0076] mTOR protein detection: About 2 x 10 6 ​One cell was added with 200 μL RIPA high-efficiency lysis solution for mechanical homogenization or ultrasonic disruption. After homogenization, 4°C, 10,000 g centrifugation for 10 minutes, the supernatant was denatured on ice. Part of the supernatant was taken for protein concentration determination. The denatured protein was subjected to protein gel electrophoresis experiment to detect the content of mTOR protein.

[0077] Cell stemness detection: Spheroid formation experiment verifies tumor cell stemness through serum-free, non-adherent three-dimensional culture system. The core process is: tumor single cell suspension (density 500-5000 cells / well) is inoculated into ultra-low adsorption culture plate, and cultured in serum-free medium added with EGF (10-20 ng / mL), bFGF (10-20 ng / mL) and B27 for 5-14 days. Ordinary tumor cells will undergo apoptosis due to adhesion dependence, while tumor stem cells with stemness will form suspended spheroids with a diameter of >75 μm. The stemness intensity is determined by the size of the spheroids.

[0078] Animal experiment: Hepatocarcinoma orthotopic tumor model: C57BL / 6 pregnant mice were bred, and the experimental mice were born within 1 day of each other. The pups were injected intraperitoneally with DEN at 12 days of age, and the development of hepatocarcinoma was induced by injecting 25% CCl4 twice a week after 45 days of birth. CCl4 injection was continued until the end of the experiment. The total experimental period was 105 days from the day of birth. At 30 days of age, the mice were divided into different experimental groups according to the order of drug administration, and the drugs were administered for 75 days. The total experimental period was 105 days from the day of birth. The liver was dissected and observed for characteristics. After fixation, HE staining was performed to determine the characteristics of tumor formation.

[0079] HE staining: The mouse liver tissue was fixed with 4% paraformaldehyde for more than 24 hours, routinely embedded with paraffin, and prepared into 4 μm thick sections. The sections were deparaffinized with xylene, hydrated with gradient ethanol (100%, 95%, 85%, 75%) and distilled water. Then the nuclei were stained with hematoxylin, and the cytoplasm was stained with eosin. The sections were dehydrated with gradient ethanol, cleared with xylene, and mounted with neutral resin. Finally, the morphological structure of the liver tissue, cell pathology, and tumor nodules were observed under a light microscope.

[0080] Example 1: Hepatocarcinoma cells HepG2 were cultured under high ammonia conditions of 5 mM NH4Cl. C-labeled glucose was used to detect the isotope change of a-KG in the cells. 13 It can be seen that the M+3 isotope of a-KG significantly increased, indicating that the reverse tricarboxylic acid cycle existed under high ammonia conditions, and cell stemness detection showed that the stemness level of the cells increased (P<0.05). Figure 1 Figure 2 ​The anti-tricarboxylic acid cycle in tumor cells directly promotes the maintenance and enhancement of tumor stemness by providing biosynthetic precursors, accumulating oncogenic metabolites, and maintaining metabolic plasticity, thereby inducing tumor development and progression. This metabolic reprogramming not only supports the self-renewal and drug resistance of tumor stem cells but also drives tumor progression through epigenetic regulation and immune escape mechanisms. Studies have found that this enhanced cell stemness through the anti-tricarboxylic acid cycle is related to the mitochondrial proteins FDXR and FDX1.

[0081] Comparative Example 1: HepG2 liver cancer cells from FDX1-KD were cultured under high ammonia conditions of 5 mM NH4Cl. 13 After labeling with C-labeled glucose, the isotopic changes of α-KG in cells were detected. Figure 1 ), and detect cell stemness ( Figure 2 This indicates a decrease in α-KG levels and a decrease in cell stemness in cells.

[0082] Comparative Example 2: HepG2 liver cancer cells from FDXR-KD were cultured under high ammonia conditions of 5 mM NH4Cl. 13 After labeling with C-labeled glucose, the isotopic changes of α-KG in cells were detected. Figure 1 ), and detect cell stemness ( Figure 2 This indicates a decrease in α-KG levels and a decrease in cell stemness in cells.

[0083] Example 2:

[0084] The site of action of ilismo is FDXR ( Figure 4 HepG2 liver cancer cells were cultured under high ammonia conditions (5 mM NH4Cl). After adding 100 nM Elesclomol, the cells were co-cultured for 3 days, and cell proliferation was assessed. Figure 5 As shown, the apoptosis rate was approximately 58.2%. Replacing with [the cell type]... 13 The isotopic changes of α-KG in cells were detected using C-labeled glucose culture medium. Figure 6 It is evident that the M+3 isotope content of α-KG is significantly decreased. Simultaneously, the cell stemming level was detected, showing a decrease in cell stemming level. Figure 7 These results indicate that Elesclomol can effectively inhibit cell proliferation and stemification by suppressing rTCA in tumor cells.

[0085] Example 3:

[0086] Reproducible experiments were performed using 9810, 97H, SW13 and A549 cell lines under high ammonia condition with 5 mM NH4Cl. After 3 days of co-culture with 100 nM elesclomol, cell proliferation was measured. As shown in Figure 5 , more than 60% of apoptosis was observed in all cell lines. This indicates that the ability of elesclomol to inhibit cell proliferation is universal.

[0087] Example 4:

[0088] HepG2 cell line was used for cell culture under high ammonia condition with 5 mM NH4Cl. First, 10 uM of copper chelator penicillamine was added for 1 day of co-culture. The medium was changed to remove free copper ions in the cells. Then, 100 nM of elesclomol was added for 3 days of co-culture. Apoptosis was measured. As shown in Figure 8 , penicillamine itself was not toxic, but after removing free copper ions in the cells, elesclomol could still cause apoptosis (52.9%). The medium containing 13 C-labeled glucose was used to detect the isotope change of a-KG in the cells. Figure 9 It can be seen that penicillamine itself does not change the M+3 isotope of a-KG, and elesclomol can still cause a significant decrease in the M+3 isotope of a-KG without copper ions. This indicates that elesclomol inhibits tumor proliferation by inhibiting rTCA in tumor cells, rather than by copper death.

[0089] Example 5:

[0090] HepG2 cell line was used for cell culture under high ammonia condition with 5 mM NH4Cl. After 3 days of co-culture with 100 nM elesclomol, the levels of lactate and LDH in the cells were measured. As shown in Figure 10 , 11 It can be seen that after long-term administration of elesclomol, the levels of lactate and LDH in the cells increased.

[0091] Example 6:

[0092] In further studies on the experiment of tumor cell dedifferentiation caused by high ammonia, we found that after long-term administration of elesclomol, the cells also activated the mTOR pathway Figure 12 , leading to cell dedifferentiation. Through cell experiments on HepG2 cells, after administration of 1 mM metformin, the mTOR pathway was inhibited by inhibiting mTOR phosphorylation.

[0093] Example 7:

[0094] HepG2 liver cancer cells were cultured under high ammonia conditions (5 mM NH4Cl). After adding 100 nM Elesclomol, the cells were co-cultured for 3 days. Simultaneously, on the third day, 1 mM metformin was administered via pulsed administration for one day, and the apoptosis rate was measured. Figure 5 As shown, the apoptosis rate increased to 66.8%. Detection of lactate and LDH levels in cells showed a significant increase compared to treatment with Elesclomol alone. Figure 10 , 11 Replace with one containing 13 The isotopic changes of α-KG in cells were detected using C-labeled glucose culture medium. Figure 6 It is evident that the M+3 isotope of α-KG decreased significantly, indicating a decrease in the degree of cell stemming. Figure 7 This indicates that the cells' ability to become stem is significantly inhibited. These findings suggest that concurrent administration of metformin via a pulsed route, while simultaneously administering Elesclomol, can significantly inhibit cell proliferation and stemification.

[0095] Example 8:

[0096] Under high ammonia conditions of 5 mM NH4Cl, cell cultures of 9810, 97H, SW13, and A549 types were used for repeated experiments. After adding 100 nM Elesclomol, cells were co-cultured for 3 days, and on the third day, a high dose of 1 mM metformin was administered via pulsed administration for one day. Cell apoptosis rate was then measured. Figure 5 As shown, the apoptosis rate increased to varying degrees in all cases. This indicates that the treatment method of administering metformin in a pulsed manner while simultaneously administering Elesclomol is quite common.

[0097] Comparative Example 3: HepG2 liver cancer cells were cultured under high ammonia conditions (5 mM NH4Cl). After co-culturing with 1 mM metformin for 3 days, the apoptosis rate was assessed. Figure 13 As shown, metformin has virtually no effect on tumor cells. This indicates that when metformin is administered alone, cell apoptosis is essentially unaffected.

[0098] Comparative Example 4: HepG2 liver cancer cells were cultured under high ammonia conditions (5 mM NH4Cl). After adding 1 mM metformin, the cells were co-cultured for 3 days. Simultaneously, on the third day, the cells were administered 100 nM Elesclomol via pulsed administration for one day, and the apoptosis rate was measured. Figure 13 As shown, the apoptosis rate was approximately 27.0%. This indicates that the order of administration of Elesclomol and metformin should not be reversed.

[0099] Comparative Example 5: HepG2 cells were cultured under high ammonia condition of 5mM NH4Cl. After 100nM elesclomol was added, the cells were co-cultured for 2 days, and on the third day, 1 mM metformin was added for 1 day. The apoptosis rate was detected. As shown in Figure 5 Table 6, the apoptosis rate was about 60.3%, and the treatment effect was slightly worse than that of Example 7, which indicated that the administration of elesclomol could not be stopped, and elesclomol was a key potent drug.

[0100] Comparative Example 6: HepG2 cells were cultured under high ammonia condition of 5mM NH4Cl. After 100nM elesclomol and 1 mM metformin were added, the cells were co-cultured for 3 days. The apoptosis rate was detected. As shown in Figure 5 Table 7, the apoptosis rate was about 62.3%. This indicated that the simultaneous administration of elesclomol and metformin was slightly worse than the pulse administration of metformin, and considering the drug toxicity and drug use cost, the overall effect of the simultaneous administration of elesclomol and metformin was not as good as that of the pulse administration of metformin.

[0101] Comparative Example 7: HepG2 cells were cultured under high ammonia condition of 5mM NH4Cl. After 100nM elesclomol was added, the cells were co-cultured for 3 days, and on the third day, other inhibitors such as rapamycin, temsirolimus, and everolimus were administered in a pulse manner for 1 day. The apoptosis rate was detected. As shown in Figure 14 Table 8, the apoptosis rate was not significantly different from that of the use of elesclomol alone. This indicated that these drugs had no significant synergistic treatment effect with elesclomol.

[0102] Example 9:

[0103] In the animal experiment of liver cancer induction, when the model mice were 30 days old, elesclomol (10 mg / kg, gavage administration) was administered daily for 75 consecutive days; metformin (200 mg / kg, intraperitoneal injection) was administered every 3 days from the 20th day of administration of elesclomol (the 50th day after the model mice were born). The experiment lasted for 105 days. The control group was administered only with physiological saline. After the mice were sacrificed, the tumor size and number were detected. It can be seen that compared with the control group, the incidence of tumors can be controlled to 4.67% (1 / 21) by this administration method, and the tumor size was significantly smaller than that of the control group. Figure 15). Liver tissue HE staining pathological evaluation showed that the liver pathological morphology of mice in the Elesclomol + pulse administration of metformin treatment group was significantly improved. Specifically, the degree of destruction of liver lobule structure was reduced, and the number and size of tumor nodules in liver tissue were significantly reduced Figure 16 .

[0104] Comparative Example 8: In the liver cancer-induced animal experiment, Elesclomol (10 mg / kg, gavage administration) was administered daily to the model mice at 30 days of age, and the administration was continued for 75 days. The experiment lasted for 105 days. After the mice were sacrificed, dissection was performed, and the size and number of tumors were detected. It was found that the tumor incidence was controlled at about 11.2%.

[0105] Comparative Example 9: In the liver cancer-induced animal experiment, metformin (200 mg / kg, intraperitoneal injection) was administered daily to the model mice at 30 days of age, and the administration was continued for 75 days. The experiment lasted for 105 days. After the mice were sacrificed, dissection was performed, and the size and number of tumors were detected. It was found that the tumor incidence was controlled at about 86.9%.

[0106] Comparative Example 10: In the liver cancer-induced animal experiment, Elesclomol (10 mg / kg, gavage administration) and metformin (200 mg / kg, intraperitoneal injection) were administered simultaneously to the model mice at 30 days of age, and the administration was continued for 75 days. The experiment lasted for 105 days. After the mice were sacrificed, dissection was performed, and it was found that the tumor incidence was controlled at about 9.34%.

[0107] Comparative Example 11: In the liver cancer-induced animal experiment, Elesclomol (10 mg / kg, gavage administration) was administered daily to the model mice at 60 days of age, and the administration was continued for 45 days. Metformin (200 mg / kg, intraperitoneal injection) was administered once every 3 days from the 10th day of Elesclomol administration (90th day after the birth of the model mice). The experiment lasted for 105 days. The control group was administered with physiological saline only, and the mice were sacrificed after dissection. It was found that the tumor incidence of the control group was 69.1%. This indicates that the combined administration of Elesclomol and metformin only has a preventive effect on tumors, and the therapeutic effect is limited.

[0108] Comparative Example 12: In the animal experiment of liver cancer induction, the model mice were administered with lenvatinib (4 mg / kg, gavage) at 30 days after birth, and the administration was continuously performed for 75 days. The experiment was performed for a total of 105 days. After the mice were sacrificed, dissection was performed, and it was found that the tumor occurrence rate was controlled to about 48.6%. When the model mice were administered with lenvatinib as a therapeutic drug at 60 days after birth, the final tumor occurrence rate of the mice was 54.2%, and the difference was not large, indicating that the therapeutic drug cannot be directly used as a preventive drug.

Claims

1. Use of elesclomol and pharmaceutically acceptable salts thereof for the preparation of a medicament for preventing cancer.

2. Use of elesclomol or pharmaceutically acceptable salts thereof in combination with metformin or pharmaceutically acceptable salts thereof for the preparation of a medicament for preventing cancer.

3. Use according to claim 2, characterized in that: The administration regimen is continuous administration of elesclomol and pulse administration of metformin, wherein the administration frequency of metformin is not less than once a week.

4. Use according to claim 1 or 2 or 3, characterized in that: The cancer includes liver cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, leukemia, lung cancer and stomach cancer.

5. Use according to claim 1 or 2 or 3, characterized in that: The medicament is a preventive medicament for high-risk population of tumor.

6. Use according to claim 5, characterized in that: The high-risk population of tumor refers to carriers of mutations in DNA repair genes, including BRCA1 / 2 mutations, Lynch syndrome, TP53 mutations; genetic tumor syndromes, including APC gene mutations, PTEN mutations; chronic infectious diseases, including HBV / HCV, HPV, Helicobacter pylori; precancerous lesion states, including Barrett's esophagus, cirrhosis, breast atypical hyperplasia, various irregular nodules; ionizing radiation exposure; chemical carcinogens exposure; long-term smokers; alcoholics; type 2 diabetes; abnormal tumor marker test.

7. A pharmaceutical composition for preventing cancer, characterized by: The effective component is composed of a first component and a second component, a) the first component is elesclomol or pharmaceutically acceptable salts thereof; b) the second component is metformin or pharmaceutically acceptable salts thereof.

8. The use according to claim 1 or 2, or the pharmaceutical composition according to claim 7, wherein: The pharmaceutically acceptable salts of elesclomol are sodium salt, potassium salt, triethylamine salt or meglumine salt; the pharmaceutically acceptable salts of metformin are hydrochloride, maleate, lactate, acetate, cyclohexylcarboxylate, nitrate, sulfate and phosphate.

9. The pharmaceutical composition of claim 7, wherein: The administration regimen is continuous administration of elesclomol and pulse administration of metformin, wherein the administration frequency of metformin is not less than once a week.

10. The pharmaceutical composition of claim 9, wherein: The starting time of pulse administration of metformin is within 2 months of the starting administration of elesclomol.

11. The pharmaceutical composition of claim 10, wherein: The starting time of pulse administration of metformin is the 20th day of the starting administration of elesclomol.

12. The pharmaceutical composition of claim 7, wherein: The administration dose of elesclomol is 2-100 mg / kg / day; the single administration dose of metformin is 10-1000 mg / kg.

13. A kit for preventing cancer, characterized by: The kit comprises elesclomol or pharmaceutically acceptable salts thereof and metformin or pharmaceutically acceptable salts thereof.

14. The kit of claim 13, wherein: Each kit contains 7 portions of elesclomol or pharmaceutically acceptable salts thereof per week, each portion having a dose of 100-5000 mg, and 1-2 portions of metformin or pharmaceutically acceptable salts thereof per week, each portion having a dose of 500-50000 mg.

15. The kit of claim 14, wherein: The elesclomol and metformin are independently packaged in the kit; the elesclomol and metformin are packaged in the kit according to the time administration arrangement; the administration regimen comprises continuous administration of elesclomol and pulse administration of metformin, wherein the administration frequency of metformin is not less than once a week.

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