Combined application of G6PD inhibitor and RSL-3 in treatment of brain glioma

Abstract

The invention particularly relates to combined application of a G6PD inhibitor and RSL-3 to treatment of brain glioma, and belongs to the technical field of medicine and biology. Specifically, the invention provides a therapeutic regimen for synergistically treating glioblastoma multiforme by using a G6PD inhibitor and RSL-3. Meanwhile, the invention provides application of G6PD in diagnosis, grading and prognosis of brain glioma.

Description

[0001] This application is a divisional application, and the parent case information is as follows: The name of the invention is combined use of G6PD inhibitors and inhibitors of new molecular points; the original application date is 2023.10.20, and the original application number is 2023113640359. Technical Field

[0002] The invention belongs to the fields of medicine and biotechnology, and specifically relates to the combined application of a G6PD inhibitor and RSL-3 in treating brain glioma. Background Art

[0003] Glioblastoma Multiform (GBM) is a WHO grade IV glioma with a median survival of only 12-15 months after diagnosis. It is the malignant tumor with the highest mortality rate in the world. In addition, the prognosis of GBM patients has not improved significantly in the past few decades, mainly due to the lack of effective treatment strategies.

[0004] The standard treatment for GBM is to first perform maximal surgical resection, followed by simultaneous radiotherapy and temozolomide (TMZ) for 6 weeks, followed by continued TMZ treatment every 4 weeks for 6 cycles. However, this treatment is only effective for a few months, and almost all GBM tumors inevitably recur after treatment. In addition to TMZ, only bevacizumab is currently approved by the FDA for the clinical treatment of recurrent GBM. However, bevacizumab can only effectively relieve symptoms and improve quality of life in the short term, and bevacizumab has no combined effect with TMZ and radiotherapy; in addition, it reduces the absorption of TMZ, cannot improve overall survival, and has significant side effects such as hypertension, venous thrombosis, and infection.

[0005] Recent studies have shown that metabolic reprogramming is one of the core characteristics of tumors. Exploring the regulatory mechanism of GBM metabolism, analyzing its biological characteristics, and finding effective molecular targets to guide clinical treatment can provide new ideas and efficient targets for cutting off GBM energy supply. Summary of the invention

[0006] In order to provide an effective diagnosis and treatment approach for brain glioma, the present invention provides the following technical solutions: A first aspect of the present invention provides a pharmaceutical composition for treating brain glioma, wherein the pharmaceutical composition comprises a first component and a second component.

[0007] The first component is a G6PD inhibitor.

[0008] The second component is a GPX4 inhibitor.

[0009] In the present invention, the term "inhibitor" refers to a substance that targets, reduces or inhibits the activity of G6PD or GPX4 protein or mRNA expression activity.

[0010] Furthermore, the G6PD inhibitor includes those that are artificially synthesized or exist in nature.

[0011] Furthermore, the G6PD inhibitors include reagents used in gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre loxP gene recombination technology, and also include compounds that can reduce the expression of G6PD.

[0012] In the present invention, G6PD refers to the gene encoding 6-phosphate glucose dehydrogenase, its transcribed mRNA and its variants, and the translated and expressed protein, and the gene ID is 2539.

[0013] In the present invention, GPX4 refers to a gene encoding glutathione peroxidase 4, its transcribed mRNA and variants thereof, and its translated and expressed protein, and its gene ID is 2879.

[0014] Furthermore, the G6PD inhibitor is G6PDi-1 or a pharmaceutically acceptable salt thereof.

[0015] Specifically, the G6PDi-1 is a reversible non-competitive 6-phosphate glucose dehydrogenase inhibitor, IC 50 It is 0.07μM, and its structural formula is:

[0016] Furthermore, the GPX4 inhibitors include those that are artificially synthesized or exist in nature.

[0017] Furthermore, the GPX4 inhibitors include reagents used in gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre loxP gene recombination technology, and also include compounds that can reduce the expression of GPX4.

[0018] Furthermore, the GPX4 inhibitor is RSL-3 or a pharmaceutically acceptable salt thereof.

[0019] In the present invention, the pharmaceutically acceptable salt refers to a salt of an active compound (such as RSL-3) and is prepared by reacting the active compound with a suitable organic or inorganic acid or acid derivative. Pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, nitrate, sodium, calcium, potassium, zinc and meglumine salts.

[0020] Certain compositions of the present invention may further comprise a pharmaceutically acceptable carrier or excipient, wherein the “pharmaceutically acceptable carrier or excipient” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary.

[0021] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient is selected from one or more of the following: a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant or an antibacterial agent.

[0022] Furthermore, the dosage forms of the first component and the second component in the pharmaceutical composition are the same or different.

[0023] Furthermore, the dosage form includes drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments or creams.

[0024] Further, the first component and the second component are administered simultaneously or sequentially. Specifically, the interval between the sequential administrations may be 0, 1, 2, 3, 4, 5, 6, 7 or more days.

[0025] Furthermore, the dosage ratio of the first component to the second component is 1:0.1-2.

[0026] Furthermore, the dosage ratio of the first component to the second component is selected from 3:1, 2:1, 1:1, 1:2, 1:3.

[0027] Furthermore, the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma.

[0028] Furthermore, the brain glioma is glioblastoma multiforme.

[0029] The pharmaceutical composition can also be used in combination with other anticancer drugs or cancer treatment methods, or the pharmaceutical composition can also contain other cancer drugs, such as: Acivicin, Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium Sodium), Bropirimine, Busulfan, Cactinomycin, Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin Hydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, Cisplatin, Cladribine, etc.

[0030] The second aspect of the present invention provides use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a product for treating brain glioma.

[0031] Furthermore, the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma.

[0032] Furthermore, the brain glioma is glioblastoma multiforme. The glioblastoma multiforme (GBM) belongs to grade IV brain glioma.

[0033] Furthermore, the product includes a test kit, a chip, a test strip, or a device, an equipment, or a computer-readable storage medium.

[0034] In the present invention, the term "treatment" refers to the process of intervening or changing a specific health state, including eliminating the cause, symptomatic treatment or supportive treatment, and does not necessarily mean curing or completely eliminating the disease, condition or symptoms of the disease or condition. Treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the severity of the symptoms / effects of an established disease.

[0035] The third aspect of the present invention provides the use of a reagent for detecting the expression of G6PD, wherein the use includes any of the following: 1) Application in the preparation of diagnostic glioblastoma products.

[0036] 2) Application in the preparation of products for grading brain gliomas.

[0037] 3) Application in the preparation of brain glioma prognosis prediction products.

[0038] Furthermore, the detection is performed on a sample from a subject, and the sample includes tissue, cells, blood, urine, saliva, mucosal samples, feces, intestinal lavage, joint fluid, cerebrospinal fluid, bile samples, respiratory secretions, and bronchoalveolar lavage fluid samples.

[0039] Furthermore, the sample is tissue or cell.

[0040] In the context of the present invention, patients or subjects are used interchangeably and refer to subjects suffering from / suspected of suffering from a disease. The disease targeted by the present invention is patients with glioma, preferably grade IV glioma, i.e. glioblastoma multiforme.

[0041] Furthermore, the product includes a test kit, a chip, a test strip, or a device, an equipment, or a computer-readable storage medium.

[0042] Furthermore, the brain glioma includes grade I, grade II, grade III, and grade IV, and the expression level of G6PD increases with the grade.

[0043] Furthermore, the prognostic indicator is the survival probability.

[0044] Furthermore, the reagent for detecting the expression level of G6PD includes a reagent for detecting the expression level of G6PD protein and / or the expression level of G6PD mRNA.

[0045] Furthermore, the reagents for detecting the expression amount of G6PD protein include reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence sorting technology and protein chip method.

[0046] Furthermore, the reagents for detecting the expression of G6PD mRNA include reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, and high-throughput sequencing platform method.

[0047] It is well known to those skilled in the art that the contents disclosed in the present invention can be specifically implemented as complete hardware, complete software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the comparison result of G6PD expression in GBM and normal brain tissue.

[0049] Figure 2 This is the comparison result of the expression level of G6PD in gliomas of different grades.

[0050] Figure 3 This is the survival curve of G6PD in GBM.

[0051] Figure 4 This is the result of using tissue chips to analyze the expression of G6PD in gliomas of different grades.

[0052] Figure 5 It is a survival curve drawn based on the results of tissue chip detection.

[0053] Figure 6 This is the result of the correlation analysis between G6PD and other genes.

[0054] Figure 7 It is the expression result of SREBP-1 and G6PD in tumor tissues of GBM patients.

[0055] Figure 8 This is a graph showing the cell number statistics of U87 cells treated with the SREBP1 inhibitor Fatostatin in combination with G6PDi-1.

[0056] Fig. 9 This is a microscopic image of U87 cells treated with the SREBP1 inhibitor Fatostatin in combination with G6PDi-1.

[0057] Fig.10 This is a graph showing the cell number statistics of U251 cells treated with the DGAT1 inhibitor A922500 in combination with G6PDi-1.

[0058] Fig.11 These are microscope images of U251 cells treated with the DGAT1 inhibitor A922500 in combination with G6PDi-1.

[0059] Fig.12 This is a graph showing the colony formation results of the DGAT1 inhibitor A922500 and G6PDi-1.

[0060] Fig.13 This is a graph showing the cell number statistics of U251 cells treated with the GPX4 inhibitor RSL-3 in combination with G6PDi-1.

[0061] Fig.14 These are microscope images of U251 cells treated with the GPX4 inhibitor RSL-3 and G6PDi-1. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0063] Example 1. Role of G6PD in the diagnosis, grading, and prognosis of GBM Through the analysis of RNAseq data of 32 solid tumors from The Cancer Genome Atlas (TCGA), it was found that the expression of G6PD in GBM was significantly higher than that in normal brain tissue ( Figure 1 ); the expression in GBM was also significantly higher than that in low-grade glioma (LGG) ( Figure 2 High expression of G6PD was significantly associated with poor survival in the GBM cohort ( Figure 3 ).

[0064] Accordingly, we further detected the G6PD protein level in tumor tissues of patients with grade I-IV gliomas using tissue microarray (TMA) (n=145). IHC staining results showed that the G6PD protein content in grade IV GBM tissues was the highest compared with grade III gliomas, grade II gliomas, and grade I gliomas ( Figure 4In addition, survival analysis showed that high levels of G6PD protein in these 145 glioma tissues were closely associated with low patient survival rates ( Figure 5 These results suggest that G6PD may be a new molecular marker for GBM and may play an important role in the occurrence and development of GBM.

[0065] Example 2: G6PDi enhances the sensitivity of cancer cells to lipid synthesis inhibitors The RNAseq data of 153 primary tumor patients in the GBM cohort in TCGA were analyzed. The results showed that the expression of G6PD was significantly positively correlated with the expression of lipid synthesis-related genes such as SREBP1, FASN, ACC, etc., and was also significantly positively correlated with the expression of genes related to maintaining intracellular redox homeostasis such as GPX4, TXN, B166, etc. ( Figure 6 ).

[0066] To evaluate the potential clinical significance of our observations, we performed a correlation analysis of the immunohistochemical staining patterns of SREBP-1 and G6PD in tumor tissues of 16 GBM patients on tissue microarray (TMA). The results showed that the expression levels of SREBP-1 and G6PD in tumor tissues of GBM patients were significantly positively correlated ( Figure 7 , some cases), among which G6PD expression was detected in 15 GBM tumor samples.

[0067] Example 3: G6PDi combined with inhibitors of new molecular points for the treatment of GBM GBM cells were treated with G6PDi-1 and a novel molecular point inhibitor to test their synergistic therapeutic effect. The drugs were purchased from MedChemExpress.

[0068] Treatment of GBM cells U87 with the SREBP1 inhibitor Fatostatin (2μM) combined with the G6PD inhibitor G6PDi-1 (5μg / ml, 10μg / ml) for 3 days produced a dose-dependent growth inhibition and promoted U87 cell death. However, treatment of U87 with Fatostatin (2μM) or G6PDi-1 (5μg / ml, 10μg / ml) alone for 3 days did not produce or only produced a weak growth inhibition effect ( Figure 8-9 , Fatostatin).

[0069] Treatment of GBM cells U251 with the DGAT1 inhibitor A922500 (DGATi, 20μg / ml) combined with the G6PD inhibitor G6PDi-1 (5μg / ml, 10μg / ml) for 4 days produced a dose-dependent growth inhibition and promoted U251 cell death. However, treatment of U251 with A922500 (20μg / ml) or G6PDi-1 (5μg / ml, 10μg / ml) alone for 4 days did not produce or only produced a weak growth inhibition effect ( Figure 10-11 A922500). After 2 weeks of treatment with the DGAT1 inhibitor A922500 (DGATi, 2μg / ml, 5μg / ml) combined with the G6PD inhibitor G6PDi-1 (5μg / ml, 10μg / ml), GBM cells U251 showed a dose-dependent growth inhibition and promoted U251 cell death ( Fig.12 , A922500 colony formation assay).

[0070] Treatment of GBM cells U251 with the GPX4 inhibitor RSL-3 (2μM) combined with the G6PD inhibitor G6PDi-1 (5μg / ml, 10μg / ml) for 4 days produced a dose-dependent growth inhibition and promoted U251 cell death. However, treatment of U251 with RSL-3 (2μM) or G6PDi-1 (5μg / ml, 10μg / ml) alone for 4 days only produced a weak growth inhibition effect ( Figure 13-14 , RSL-3).

Claims

1. A pharmaceutical composition for treating brain glioma, characterized in that: The pharmaceutical composition comprises a first component and a second component; The first component is a G6PD inhibitor; The second component is a GPX4 inhibitor; Preferably, the G6PD inhibitor includes artificially synthesized or naturally occurring ones; Preferably, the G6PD inhibitor includes reagents used in gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre loxP gene recombination technology, and also includes compounds that can reduce the expression of G6PD; Preferably, the G6PD inhibitor is G6PDi-1 or a pharmaceutically acceptable salt thereof; Preferably, the GPX4 inhibitor includes artificially synthesized or naturally occurring ones; Preferably, the GPX4 inhibitor includes reagents used in gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre loxP gene recombination technology, and also includes compounds that can reduce the expression of GPX4; Preferably, the GPX4 inhibitor is RSL-3 or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient is selected from one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH regulator, antioxidant or antibacterial agent.

3. The pharmaceutical composition according to claim 1, characterized in that The dosage forms of the first component and the second component in the pharmaceutical composition are the same or different; Preferably, the dosage form includes drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments or creams.

4. The pharmaceutical composition according to claim 1, characterized in that The first component and the second component are administered simultaneously or sequentially, specifically, the interval between the sequential administrations may be 0, 1, 2, 3, 4, 5, 6, 7 or more days; Preferably, the dosage ratio of the first component to the second component is 1:0.1-2; Preferably, the dosage ratio of the first component to the second component is selected from 3:1, 2:1, 1:1, 1:2, 1:

3.

5. The pharmaceutical composition according to claim 1, characterized in that The brain glioma includes grade I, grade II, grade III, and grade IV brain glioma; Preferably, the brain glioma is glioblastoma multiforme.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a product for treating brain glioma; Preferably, the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma; Preferably, the brain glioma is glioblastoma multiforme; Preferably, the product includes a kit, a chip, a test strip, or a device, an apparatus, or a computer-readable storage medium.

7. Use of a reagent for detecting the expression of G6PD, characterized in that: The application includes any of the following: 1) Application in the preparation of diagnostic glioblastoma products; 2) Application in the preparation of products for grading brain gliomas; 3) Application in the preparation of brain glioma prognosis prediction products.

8. The use according to claim 7, characterized in that: The test is performed on a sample from a subject, and the sample includes tissue, cells, blood, urine, saliva, mucosal samples, feces, intestinal lavage, joint fluid, cerebrospinal fluid, bile samples, respiratory secretions, and bronchoalveolar lavage fluid samples; Preferably, the sample is tissue or cell; Preferably, the product includes a kit, a chip, a test strip, or a device, an apparatus, or a computer-readable storage medium.

9. The use according to claim 7, characterized in that: The brain glioma includes grade I, grade II, grade III, and grade IV, and the expression of G6PD increases with the grade; Preferably, the prognostic indicator is the survival probability.

10. The use according to claim 7, characterized in that The reagent for detecting the expression of G6PD includes a reagent for detecting the expression of G6PD protein and / or the expression of G6PD mRNA; Preferably, the reagents for detecting the expression of G6PD protein include reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence sorting technology and protein chip method; Preferably, the reagents for detecting the expression of G6PD mRNA include reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, and high-throughput sequencing platform method.

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