Application of FR054 in reversing temozolomide drug resistance of glioblastoma

Abstract

The invention discloses application of FR054 in reversing temozolomide drug resistance of glioblastoma, FR054 is used as a sensitizer of temozolomide, and the sensitivity of temozolomide to tumor cells and the killing effect of temozolomide to GBM cells are enhanced by targeted inhibition of a key metabolic enzyme PGM3 in an HBP pathway.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of FR054 in reversing temozolomide resistance in glioblastoma. Background Art

[0002] Glioblastoma (GBM) is one of the most common primary malignant brain tumors in adults, characterized by high invasiveness, rapid growth, and significant genetic heterogeneity. According to the World Health Organization (WHO) classification, GBM is classified as a grade IV astroblastoma. Its clinical presentation is often non-specific, resulting in most patients being diagnosed at an advanced stage. Despite recent advances in treatment options such as surgery, radiotherapy, and chemotherapy, the prognosis for patients remains poor. Under current standard treatment options, the median survival for GBM patients is approximately 15 months, and the five-year survival rate is less than 10%.

[0003] The current standard first-line treatment for newly diagnosed GBM is the Stupp regimen, which involves maximal safe surgical resection of the tumor, followed by postoperative radiation therapy and concurrent and adjuvant temozolomide (TMZ) chemotherapy. Established in 2005, this regimen remains the primary treatment strategy widely used in clinical practice. However, due to the highly invasive nature of GBM, complete resection is virtually impossible, making postoperative adjuvant therapy crucial for controlling tumor recurrence.

[0004] TMZ is a second-generation oral alkylating agent. It hydrolyzes to form methyltriazene imidazole amide (MTIC), which further decomposes into active substances such as diazomethane, causing methylation of DNA guanine O6 and N7 sites and adenine O3 sites, thereby activating the DNA mismatch repair system and ultimately inducing double-strand breaks, cell cycle arrest, and apoptosis. Although TMZ has some clinical efficacy, most patients still develop drug resistance after treatment, leading to disease recurrence. Therefore, how to effectively overcome TMZ resistance has become a key challenge in improving the treatment of GBM. Currently, there are no clinically available drugs that effectively sensitize GBM cells to TMZ treatment.

[0005] In recent years, researchers have begun to focus on the role of tumor metabolic regulation in the development of drug resistance. In particular, glucose metabolism pathways such as the hexosamine biosynthesis pathway (HBP) have been found to be closely associated with tumor proliferation, metastasis, and treatment resistance in various cancers. HBP is a key branch of glucose metabolism, accounting for approximately 2%-3% of total glucose uptake. This pathway, which starts with fructose-6-phosphate and glutamine and undergoes a series of enzymatic reactions, ultimately produces uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is not only an important substrate for protein O-GlcNAc glycosylation, but also participates in various biological processes, including cellular structure maintenance, signal transduction, and immune evasion.

[0006] PGM3 is a key metabolic enzyme in the HBP pathway. Regarding the role of PGM3 in GBM cells, Su H and Zhong et al. published a paper titled "Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth," reporting that PGM3 is a promising target for the treatment of glioblastoma (GBM). Inhibiting PGM3 can disrupt the positive feedback regulation between SREBP-1 activation and hexosamine synthesis, thereby effectively killing glioblastoma (GBM) cells. This study only revealed the role of PGM3 as an oncogene in mediating GBM cell proliferation, and did not report the role and mechanism of PGM3 in GBM cells' resistance to TMZ or sensitization to TMZ treatment.

[0007] FR054 is a novel competitive inhibitor of the HBP enzyme PGM3. Its chemical name is (5H-Pyrano[3,2-d]oxazole-6,7-diol, 5-[(acetyloxy)methyl]-3a,6,7,7a-tetrahydro-2-methyl-, diacetate (ester), (3aR,5R,6S,7R,7aR)-]. FR054 was initially developed for the treatment of breast cancer, but its mechanism of action and potential applications suggest it may be useful in the treatment of a variety of cancers. As a PGM3 inhibitor, FR054 reduces the formation of UDP-GlcNAc by interfering with a key step in the HBP. This not only affects the N- and O-glycosylation levels of cell surface proteins but may also influence various cellular processes, such as proliferation, apoptosis, stress response, and signal transduction, by regulating protein O-GlcNAc glycosylation modifications. Studies have shown that FR054 can induce growth arrest and cell death in breast cancer, pancreatic cancer, and pancreatic ductal adenocarcinoma cell lines in vitro and reduce the N- and O-glycosylation levels of these cells. Furthermore, FR054 can activate the unfolded protein response and increase the accumulation of intracellular reactive oxygen species (ROS). In a pancreatic cancer model, the combination of FR054 and gemcitabine can enhance the effects of the latter and improve treatment efficiency. In the field of glioma, there are currently no reports of the use of FR054 to reverse GBM cell resistance to TMZ treatment.

[0008] Based on the above research background, no literature has yet proposed a potential relationship between PGM3 and TMZ resistance, nor has there been any research or application of FR054 for reversing TMZ resistance in GBM. There is no in vivo or in vitro experimental data on the synergistic anti-tumor effect of FR054 combined with TMZ in GBM models, nor has the mechanism of how FR054 affects TMZ sensitivity by regulating HBP and its downstream O-GlcNAc modification been elucidated. Summary of the Invention

[0009] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a method for reversing temozolomide resistance in glioblastoma using FR054.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides use of an HBP pathway inhibitor in the preparation of a temozolomide sensitizer.

[0011] The HBP pathway inhibitor in the present invention refers to a substance that can block the key steps in the HBP pathway to reduce the generation of UDP-GlcNAc, thereby affecting the level of protein O-GlcNAc modification.

[0012] In some embodiments, the HBP pathway inhibitor is a PGM3 inhibitor.

[0013] In some embodiments, the PGM3 inhibitor comprises at least one of a substance that inhibits PGM3 activity, a substance that degrades PGM3, and a substance that reduces the expression level of PGM3.

[0014] In some embodiments, the PGM3 inhibitor is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor, or a small hairpin RNA (shRNA).

[0015] In some embodiments, the PGM3 inhibitor is FR054 or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof.

[0016] The second aspect of the present invention provides a combination drug comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is an HBP pathway inhibitor and the second active ingredient is temozolomide.

[0017] In some embodiments, the HBP pathway inhibitor is a PGM3 inhibitor.

[0018] In some embodiments, the PGM3 inhibitor comprises at least one of a substance that inhibits PGM3 activity, a substance that degrades PGM3, and a substance that reduces the expression level of PGM3.

[0019] In some embodiments, the PGM3 inhibitor is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor, or a small hairpin RNA (shRNA).

[0020] In some embodiments, the PGM3 inhibitor is FR054 or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof.

[0021] In some embodiments, the combination comprises a therapeutically effective amount of temozolomide and a therapeutically effective amount of FR054.

[0022] The combination can be used to treat tumors, particularly glioblastoma. In one embodiment, the combination containing a therapeutically effective amount of temozolomide and a therapeutically effective amount of FR054 is formulated and administered in a manner that is conducive to pharmaceutical use, taking into account the individual patient's clinical condition, delivery site, administration method, administration schedule, and other factors known to the physician. Therefore, the "effective amount" for the purposes of this article is determined by these considerations.

[0023] In some embodiments, the dosage of FR054 is 100-150 mg per kilogram of body weight per day, and the dosage of temozolomide is 15-25 mg per kilogram of body weight per day.

[0024] In some embodiments, the dosage of FR054 is 120 mg per kilogram of body weight per day, and the dosage of temozolomide is 20 mg per kilogram of body weight per day.

[0025] In some embodiments, the first active ingredient and the second active ingredient are in the same formulation unit; or the first active ingredient and the second active ingredient are in different formulation units.

[0026] In some embodiments, the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially.

[0027] In some embodiments, the dosage form of the combination drug is a parenteral dosage form or a parenteral dosage form.

[0028] In some embodiments, the dosage form of the combination drug is any one of powder, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral solutions, ointments, emulsions, and irrigation solutions.

[0029] In some embodiments, the combination drug further comprises a pharmaceutically acceptable excipient.

[0030] In some embodiments, the excipients further include at least one of a filler, a diluent, a disintegrant, a binder, a lubricant, a glidant, a surfactant, a solvent, a flavoring agent, and a preservative.

[0031] In some embodiments, the filler or diluent includes sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0032] In some embodiments, the lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silicon dioxide; micropowder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0033] In some embodiments, the binder includes distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, ethyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, and compounds similar to the above excipients.

[0034] In some embodiments, the disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0035] In some embodiments, the surfactant includes Tween, sodium lauryl sulfate, sodium stearate sulfonate, and the like.

[0036] In some embodiments, the antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid, and the like.

[0037] In some embodiments, the preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0038] In some embodiments, the flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, mint oil, menthol, mint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0039] The third aspect of the present invention provides use of the combined drug of the second aspect in the preparation of a drug for treating tumors.

[0040] In some embodiments, the tumor is a glioblastoma.

[0041] In some embodiments, the glioma is a temozolomide-resistant glioblastoma.

[0042] In some embodiments, the combination drug treats tumors through at least one of the following pathways: Increase iron levels in tumor cells; Increase ROS levels in tumor cells; Induces ferroptosis in tumor cells; Reduce GSH content in tumor cells; Reduce GPX4 levels in tumor cells; Increases HMOX1 levels in tumor cells.

[0043] The beneficial effects of the present invention are: The present invention uses FR054 as a sensitizer for temozolomide, and enhances the sensitivity of temozolomide to tumor cells and the killing effect of temozolomide on GBM cells by targeted inhibition of PGM3, a key metabolic enzyme in the HBP pathway.

[0044] By restoring the sensitivity of temozolomide, this invention is expected to significantly increase the success rate of first-line treatment, prolong patients' progression-free survival and overall survival, and improve their quality of life. Temozolomide is a relatively inexpensive and widely used standard treatment drug. By enhancing its efficacy with FR054, it eliminates the need for expensive new drugs or complex treatment regimens, helping to reduce overall treatment costs and improve the cost-effectiveness of treatment. It is particularly suitable for medical institutions in resource-limited areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The cell viability and IC values ​​of each tumor cell before and after TMZ treatment in Example 1 of the present invention are shown in Table 1. 50 result.

[0046] Figure 2 The cell viability and IC values ​​of GBM cell lines at different TMZ concentrations after adding FR054 in Example 1 of the present invention are shown in Table 1. 50 .

[0047] Figure 3 These are the results of GBM cell plate cloning experiments in different treatment groups in Example 1 of the present invention.

[0048] Figure 4 The organoid morphological changes under light microscopy in Example 2 of the present invention are shown when DMSO is added, TMZ or FR054 is used alone, and TMZ and FR054 are used in combination.

[0049] Figure 5 The staining results of Example 2 of the present invention, in which DMSO was added, TMZ or FR054 was used alone, and TMZ and FR054 were used in combination, were analyzed under a fluorescence microscope at 100 times magnification and under the FITC excitation / emission channel conditions, and the green fluorescence positive rate was calculated using imageJ.

[0050] Figure 6 These are the in vivo bioluminescence imaging results of nude mice in each treatment group every 7 days in Example 3 of the present invention.

[0051] Figure 7 The fluorescence signal intensity of the tumor in nude mice in each treatment group after treatment in Example 3 of the present invention was obtained using the Wilcoxon rank sum test.

[0052] Figure 8 This is the survival curve of nude mice in each treatment group in Example 3 of the present invention. *p<0.05, ns:p>0.05.

[0053] Figure 9 The figure shows the changes in body weight of nude mice in each treatment group during treatment in Example 3 of the present invention.

[0054] Figure 10 Fluorescence microscopy images and quantitative analysis of ferrous ion levels in GBM cells of each treatment group detected by FerroOrange staining in Example 4 of the present invention (72 hours of treatment), n = 3; ns: p>0.05.

[0055] Figure 11 These are the FITC channel fluorescence images and mean fluorescence intensity quantitative results of DCFH-DA staining to detect ROS levels in GBM cells in each treatment group in Example 4 of the present invention (72 hours of treatment), n = 3; ns: p>0.05.

[0056] Figure 12 The PTGS2 mRNA expression levels in GBM cells of each treatment group were detected by qPCR in Example 4 of the present invention, with ACTB as the internal reference and the DMSO group as the control for normalization.

[0057] Figure 13 The GSH detection kit in Example 4 of the present invention was used to measure the glutathione content in GBM cells of each treatment group.

[0058] Figure 14 The Western blot assay in Example 4 of the present invention detected the expression levels of ferroptosis-related proteins HMOX1 and GPX4 in GBM cells. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below by way of specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the examples are all commercially available or can be obtained by conventional methods. Unless otherwise specified, all experiments or testing methods are conventional methods in the art.

[0060] Example 1 Effect of FR054 combined with TMZ on the therapeutic effect of GBM cells GBM cell lines and GBM TMZ-resistant cell lines were used to test the combined efficacy of FR054 and TMZ. First, a TMZ-resistant GBM cell line was constructed using the drug concentration escalation method.

[0061] 1. Construction of TMZ-resistant tumor cell lines GBM cell lines U87-MG and A172 (purchased from American Type Culture Collection, ATCC) were selected to test the cell viability and IC of each tumor cell line. 50 The method was based on the applicant's previous publication J Exp Clin Cancer Res 2023 Vol. 42 Issue 1 Pages 285. The tumor cell lines were plated on a 10 cm culture dish at a density of approximately 70%-80%. Then, a TMZ solution containing the IC values ​​of each cell line was added. 50 One tenth (10% IC 50 The cells were cultured in DMEM medium with 10% FBS and double-antibody (100 U / mL penicillin and 100 μg / mL streptomycin) for 72 h, and then replaced with drug-free medium for 72 h. The culture was then repeated with drug-containing and drug-free medium. The TMZ concentration in the drug-containing medium was gradually increased (20% IC 50 、40%IC 50 、60%IC 50 , 80% IC 50 、100%IC 50 , 200%IC 50 ), the treatment time was 6 months. After 6 months, the cell viability and TMZIC 50 The tumor cell lines treated with U87-MG and A172 were designated as U87-TR and A172-TR respectively.

[0062] In this embodiment, the tumor cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0063] Cell viability and IC of each GBM cell line before and after TMZ treatment 50 The results are as follows Figure 1 As shown, the IC values ​​of each tumor cell line after TMZ treatment were 50 Both reached more than twice the level before TMZ treatment, indicating that the TMZ-resistant tumor cell line was successfully constructed.

[0064] 2. Testing the efficacy of FR054 combined with TMZ on GBM cells The above-mentioned successfully constructed TMZ-resistant GBM cell lines U87-TR, A172-TR and original cell lines U87 and A172 were used to test the therapeutic effects of TMZ alone, FR054 alone, and TMZ combined with FR054.

[0065] GBM cell lines U87 and A172 were cultured in culture medium (DMEM+10% FBS+double antibodies (100 U / mL penicillin and 100 μg / mL streptomycin)) containing different concentrations of TMZ (0µM, 100µM, 200µM, 400µM, 800µM, 1600µM, 3200µM) and different concentrations of FR054 (0µM, 25µM, 50µM). After 72 hours of culture, CCK8 drug sensitivity assay was performed (the detection method was referred to the applicant's previous publication J Exp Clin Cancer Res 2023 Vol. 42 Issue 1 Pages 285. PMCID: PMC10612313 DOI: 10.1186 / s13046-023-02843-6). TMZ-resistant GBM cell lines U87-TR and A172-TR were cultured in culture medium (DMEM medium + 10% FBS + double antibody (100 U / mL penicillin and 100 μg / mL streptomycin)) containing different concentrations of TMZ (0µM, 100µM, 200µM, 400µM, 800µM, 1600µM, 3200µM) and different concentrations of FR054 (0µM, 50µM, 75µM). After 72 hours of culture, CCK8 drug sensitivity test was performed. The results are shown in Figure 3. Figure 2 IC values ​​of TMZ in each cell line after adding FR054 50 All of them showed a significant decrease, indicating that FR054 can enhance the sensitivity of GBM cell lines to TMZ.

[0066] To further evaluate the effect of FR054 combined with temozolomide (TMZ) on the clonogenic ability of glioblastoma (GBM) cells, the present invention used a plate clone formation assay to perform in vitro functional verification.

[0067] The specific operations are as follows: GBM cell lines U87 and A172 were seeded in 6-well plates at a seeding density of approximately 1000 cells per well; for TMZ-resistant cell lines U87-TR and A172-TR, the seeding density was the same. After plating, culture medium containing the following drug combinations (DMEM medium + 10% FBS + dual antibodies (100 U / mL penicillin and 100 μg / mL streptomycin)) was added for treatment: control group: DMSO; TMZ single-drug group: TMZ 200 μM (for U87 and A172) or TMZ 600 μM (for U87-TR and A172-TR); FR054 single-drug group: FR054 25 μM (for U87 and A172) or FR054 50 μM (for U87-TR and A172-TR); combination drug group: TMZ 200 μM + FR054 25 μM (for U87 and A172) or TMZ 600 μM + FR054 50 μM (for U87-TR and A172-TR).

[0068] The cells were cultured in a 37°C, 5% CO2 incubator for 14 days, with fresh drug-containing culture medium replaced every 72 hours. Following incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet. Microscopic images were taken to document the cell colony formation in each group.

[0069] Subsequently, ImageJ image analysis software was used to perform statistical analysis on the number of clones. Figure 3 The results showed that compared with the single-drug group and the control group, the combination of FR054 and TMZ significantly inhibited the cloning ability of GBM cells, indicating that the two drugs have a synergistic effect in inhibiting the long-term survival ability of tumor cells.

[0070] Example 2 Effect of FR054 combined with TMZ on the therapeutic efficacy of organoid models derived from tumor patients Organoids from five glioma patients at Nanfang Hospital of Southern Medical University were selected. The organoid construction method referred to the applicant's previous publication J Exp Clin Cancer Res 2023 Vol. 42 Issue 1 Pages 285. PMCID: PMC10612313 DOI: 10.1186 / s13046-023-02843-6. These five organoids were named PDO01, PDO02, PDO03, PDO04, and PDO05 respectively. These five organoids were separated into single organoids using a pipette, seeded into 24-well plates, and then cultured for 96 h using culture medium (DMEM medium + 10% FBS + double antibody (100 U / mL penicillin and 100 μg / mL streptomycin)) containing DMSO, 1000μM TMZ, 50μM FR054, and 1000μM TMZ+50μM FR054, respectively. The culture conditions were based on the applicant's previous publication (J Exp Clin Cancer Res 2023 Vol. 42 Issue 1 Pages 285. PMCID: PMC10612313 DOI: 10.1186 / s13046-023-02843-6). The images were then photographed under a 100x light microscope. The results are shown in Figure 6. Figure 4 Under combined drug treatment conditions, GBM organoids showed significant morphological changes compared to the single drug treatment group, indicating that the combination of the two drugs has a more significant biological effect in regulating the structural characteristics of organoids.

[0071] Furthermore, SYTOX Green dead cell staining was used for staining analysis of the drug-treated organoids. The specific steps were as follows: the organoids in a 24-well plate were washed three times with phosphate-buffered saline (PBS); SYTOX Green dye was dissolved in PBS at a ratio of 1:30,000 to prepare a working staining solution; this working staining solution was added to the 24-well plate and incubated for 30 minutes in a dark, 37°C, 5% CO₂ environment; after incubation, the organoids were washed three times with PBS; and the staining results were observed and images were captured under a fluorescence microscope using a 100x magnification lens and FITC excitation / emission channels. The green fluorescence positive rate was calculated using ImageJ.

[0072] The results are as follows Figure 5 As shown in the data, the SYTOX Green positivity rate of the FR054 and TMZ combination group was significantly higher than that of the single-drug group and the DMSO group, which indicates that the combination of the two drugs has a more significant killing effect on organoids than the single-drug group and the DMSO group, and the combination of FR054 and TMZ has a synergistic inhibitory effect on GBM organoids.

[0073] Example 3 Effect of FR054 combined with TMZ on the therapeutic efficacy of the orthotopic intracranial tumor model in nude mice 1. Construction of luciferase-labeled U87 cells (hereinafter referred to as U87-LUC) U87 cells were transfected with the LV-U6-Luci17-T2A-NEO plasmid purchased from Genema (Suzhou) Co., Ltd. according to the manufacturer's instructions. U87 cells in logarithmic growth phase were seeded in complete medium (DMEM + 10% FBS + double-antibody (100 U / mL penicillin and 100 μg / mL streptomycin)) and cultured in a 37°C, 5% CO2 incubator until confluency reached 60%–70%. The LV-U6-Luci17-T2A-NEO plasmid was then transfected into the U87 cells using Lipofectamine 3000 according to the manufacturer's recommended ratio. Specifically, the appropriate amount of plasmid DNA and transfection reagent mixture was added to fresh antibiotic-free complete medium, gently mixed, and then plated. Eight hours after transfection, the medium was replaced with fresh complete medium, and the cells were cultured at 37°C, 5% CO2. G418 was added at a selection concentration of 500 μM 48 hours after transfection and selection continued for 7 days. During this period, the culture medium containing selective antibiotics was regularly replaced, and cell viability was observed. After 7 days of screening, the cells were replaced with DMEM containing 0.5 mg / mL potassium luciferin for 5 minutes. The cell fluorescence signal was observed on a live imaging device to confirm transfection efficiency before use in subsequent experiments.

[0074] 2. Intracranial transplantation of U87-LUC cells to establish a human glioblastoma nude mouse model The successfully constructed U87-LUC cells were implanted into the skull of nude mice. The specific process was as follows: the tumor cells in the logarithmic growth phase were digested with trypsin, washed twice with PBS, and finally resuspended in PBS to adjust the concentration to about 1×10 5 Cells per microliter. Nude mice (all 4-5 week old, BALB / c strain) were purchased from Beijing Sibeifu Biotechnology Co., Ltd.) were anesthetized with 0.1% phenobarbital. 100 μL of anesthetic was injected per 20 g nude mouse until the mouse lost its response to external stimuli. The anesthetized nude mouse was secured in a stereotaxic apparatus with its head facing the operating area, and the scalp was cleaned with disinfectant. A microdrill was used to carefully drill the skull at predetermined coordinates (1 mm anterior to bregma and 1.5 mm lateral to the right) without damaging brain tissue. Using a microsyringe, 10 μL of the pre-prepared tumor cell suspension was slowly injected to a predetermined depth of 1.5 mm. The injection rate was controlled at approximately 1 μL / min. After completion, the needle was held in place for several minutes before being slowly withdrawn to prevent backflow of the fluid. Ten days after tumor formation, the nude mice were observed using a small animal imaging device to confirm tumor development.

[0075] 3. Testing the efficacy of FR054 combined with TMZ in nude mouse models The nude mice with established tumors were randomly divided into five different treatment groups: negative control group (NC), temozolomide alone group (TMZ, 20 mg / kg qd), high-dose FR054 monotherapy group (FR054high, 120 mg / kg bid), temozolomide plus high-dose FR054 combination therapy group (TMZ+FR054 high ), and temozolomide plus low-dose FR054 (60 mg / kg bid) (TMZ+FR054 low ) combined treatment group, 6 mice per group. All drugs were administered by intraperitoneal injection using 50% PGE300 and 50% saline as solvents. Each cycle was 5 days of treatment and 2 days of injection cessation, for a total of two cycles. Every 7 days, in vivo imaging was performed on each group of nude mice to monitor the development of intracranial tumors. Figure 6 At the same time, the intracranial fluorescence signal intensity after treatment was recorded and analyzed ( Figure 7 ).

[0076] The results showed that the fluorescence signal intensity of nude mice treated with either FR054 or temozolomide alone was significantly lower than that of the negative control group (p<0.001). It is worth noting that in the group treated with temozolomide combined with high-dose FR054, the tumor fluorescence signal intensity observed was significantly lower than that of the group treated with temozolomide alone (p=0.08), while no significant difference was observed between the group treated with temozolomide combined with low-dose FR054 and the group treated with temozolomide alone. In addition, the observation of the survival period of nude mice showed that the trend of the results was consistent with the changes in the fluorescence signal intensity mentioned above ( Figure 8 This indicates that either FR054 or temozolomide alone can effectively inhibit tumor growth, while the combination of high-dose FR054 and temozolomide showed a more significant therapeutic effect. In contrast, the combination of low-dose FR054 and temozolomide failed to further enhance the therapeutic effect. Throughout the treatment process, the body weight of nude mice was regularly monitored ( Figure 9 ), no significant body weight differences were observed among the groups, suggesting that the drug regimen used had no significant adverse effects on the overall health of the nude mice.

[0077] Example 4: Combination of FR054 and TMZ induces increased ferroptosis in GBM cells To further verify the mechanism of action of the combination of FR054 and temozolomide (TMZ), the present invention conducted the following experiments.

[0078] 1. Detection of iron ion and ROS levels FerroOrange and DCFH-DA reagents were used to measure intracellular iron and reactive oxygen species (ROS) levels, respectively. The specific steps were as follows: GBM cell lines U87 and A172, as well as drug-resistant cell lines U87-TR and A172-TR, were seeded in six-well plates. After the cells attached, they were cultured with medium (DMEM medium + 10% FBS + dual antibodies (100 U / mL penicillin and 100 μg / mL streptomycin)) containing DMSO (control), TMZ alone, FR054 alone, or the combination of TMZ and FR054. The cells were incubated at 37°C in a 5% CO2 incubator. After 72 hours of culture, the medium was discarded and the cells were washed twice with PBS. For iron detection, FerroOrange dye was used according to the manufacturer's instructions, and the cells were observed and photographed under a fluorescence microscope. For ROS detection, DCFH-DA dye was used according to the manufacturer's instructions, and the cells were observed and photographed under a fluorescence microscope. The mean fluorescence intensity was calculated using ImageJ. The results showed that compared with the control group and other treatment groups, the combination of TMZ and FR054 significantly increased the intracellular iron ion level and ROS level ( Figure 10-11 ).

[0079] 2. Detection of PTGS2 mRNA expression level Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of PTGS2 mRNA as a marker of ferroptosis. The specific steps were as follows: After treating the cells for 72 hours according to the above method, total RNA was extracted and reverse transcribed into cDNA. Specific primers were used for qRT-PCR reaction, and β-actin was used as the internal reference gene. The relative expression level was calculated and statistical analysis was performed. The results showed that in the combination treatment group, PTGS2 mRNA expression was significantly upregulated, indicating that the combination treatment induced ferroptosis ( Figure 12 ).

[0080] 3. Glutathione (GSH) content detection: Use a GSH detection kit to quantitatively detect intracellular GSH content. The specific steps are as follows: After treating cells for 72 hours according to the above method, collect cell lysate. According to the kit instructions, measure GSH content and calculate the mean and standard deviation of each group. The results showed that compared with the control group and other treatment groups, the combined drug significantly reduced the GSH content in GBM cells ( Figure 13 ), further supporting the occurrence of ferroptosis.

[0081] 4. Detection of GPX4 and HMOX1 protein expression levels As increasing research indicates that GPX4 and HMOX1 proteins play important roles in ferroptosis induction, we examined their expression levels by Western blotting. The specific steps were as follows: After treating cells for 48 hours as described above, cell lysates were collected and SDS-PAGE samples were prepared. Electrophoresis was performed, the samples were transferred to membranes, and immunoblotting was performed using GPX4- and HMOX1-specific antibodies. Band intensities were detected and quantified using a chemiluminescence imaging system.

[0082] The results showed that in the combination group, GPX4 expression was significantly downregulated, while HMOX1 expression was significantly upregulated ( Figure 14 ), indicating that the combined treatment promoted ferroptosis by regulating these key proteins.

[0083] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of HBP pathway inhibitors in the preparation of temozolomide sensitizers.

2. The use according to claim 1, characterized in that The HBP pathway inhibitor is a PGM3 inhibitor.

3. The use according to claim 2, characterized in that The PGM3 inhibitor is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor or a small hairpin RNA (shRNA).

4. The use according to claim 2, characterized in that The PGM3 inhibitor is FR054 or a salt, stereoisomer, tautomer or isotope-labeled compound thereof.

5. A combination drug comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is an HBP pathway inhibitor and the second active ingredient is temozolomide.

6. The combined drug according to claim 5, characterized in that The HBP pathway inhibitor is a PGM3 inhibitor.

7. The combined drug according to claim 6, characterized in that The PGM3 inhibitor is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA, a small molecule inhibitor or a small hairpin RNA.

8. The combined drug according to claim 6, characterized in that The PGM3 inhibitor is FR054 or a salt, stereoisomer, tautomer or isotope-labeled compound thereof.

9. The combined drug according to claim 5, characterized in that The first active ingredient and the second active ingredient are in the same preparation unit; or the first active ingredient and the second active ingredient are in different preparation units.

10. Use of the combined drug according to any one of claims 5 to 9 in the preparation of a drug for treating tumors.

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